AGENT ENHANCING ANTITUMOR EFFECT USING PYRAZOLO[3,4-d]PYRIMIDINE COMPOUND

ABSTRACT

To provide a method for treating cancer using a pyrazolo[3,4-d]pyrimidine compound or a salt thereof. The present invention provides an antitumor agent comprising a pyrazolo[3,4-d]pyrimidine compound of formula (I) wherein X, Y, Z1, Z2, Z3, Z4, W, n, R1, R2, and R3 have meanings as defined in the present specification, or a salt thereof and other antitumor agent(s) for combined administration.

FIELD OF THE INVENTION

The present invention relates to an antitumor agent comprising apyrazolo[3,4-d]pyrimidine compound or a salt thereof and other antitumoragent(s) in combination, and an agent enhancing an antitumor effect ofother antitumor agent(s).

BACKGROUND OF THE INVENTION

HER2 (also called ErbB2) is a receptor-type tyrosine kinase belonging tothe ErbB family.

HER2 is considered as a gene responsible for cancer (Non PatentLiterature 1), and HER2 gene amplification, mutation, overexpression,and the like have been reported on various cancers (Non PatentLiterature 2). It has been reported that the survival, growth signals,etc. of the cancer cells are increased by the activation of the signaltransmission of HER2 and its downstream pathway in cancer cells havingsuch HER2 gene abnormality or overexpression (Non Patent Literatures 3and 4).

Thus, an inhibitor capable of controlling the kinase activity of HER2presumably exerts antitumor effect by inhibiting the signal transductionof HER2 and its downstream pathway in cancer cells, and is thereforeconsidered to be useful as a therapeutic drug for cancer.

There are a large number of reports on antitumor effects brought aboutby combinations of HER2 inhibitors and other antitumor agents. Forexample, combined use of trastuzumab with paclitaxel (Non PatentLiterature 5), paclitaxel/platinum compound (Non Patent Literature 6),or the like has been reported. Also, combined use of lapatinib withcapecitabine (Non Patent Literature 7), 5-FU (Non Patent Literature 8),S-1 (Non Patent Literature 9), or the like has been reported. However,for example, lapatinib is known to exhibit high inhibitory activityagainst EGFR (epidermal growth factor receptor), in addition to HER2,and might thus cause adverse effects due to the inhibition of thesignaling pathway of EGFR.

Meanwhile, the present applicant has previously filed a patentapplication by finding that a compound having pyrazolo[3,4-d]pyrimidineas a basic structure has excellent inhibitory activity against HER2 andkinase selectivity for HER2.

CITATION LIST Non Patent Literature

-   Non Patent Literature 1: Oncogene, 26, p. 6469-6487 (2007)-   Non Patent Literature 2: Cancer Treat. Rev., 40, p. 770-780 (2014)-   Non Patent Literature 3: Genes Cancer, 4, p. 187-195 (2013)-   Non Patent Literature 4: Oncogene, 19, p. 1647-1656 (2000)-   Non Patent Literature 5: N. Engl. J. Med., 344, p. 783-792 (2001)-   Non Patent Literature 6: J. Natl. Cancer Inst., 96, p. 759-769    (2004)-   Non Patent Literature 7: Breast Cancer, 21, p. 677-683 (2014)-   Non Patent Literature 8: Oncol. Rep., 27, p. 1639-1645 (2012)-   Non Patent Literature 9: Mol. Cancer Ther., 9, 1198-1207 (2010)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method for treatingcancer using a pyrazolo[3,4-d]pyrimidine compound or a salt thereofwhich exhibits an excellent antitumor effect while preventing adverseeffects.

Means for Solving the Problem

The present inventors have conducted diligent studies to attain theobject and consequently completed the present invention by finding thatcombined use of a compound of formula (I) below havingpyrazolo[3,4-d]pyrimidine as a basic structure or a salt thereof withother compounds having an antitumor effect (other antitumor agents) moreenhances the antitumor effect than use of each of these compounds alone,but does not enhance toxicity.

The present invention provides the following [1] to [36]:

[1] An antitumor agent, wherein a pyrazolo[3,4-d]pyrimidine compound ofthe following formula (I) or a salt thereof and other antitumor agent(s)are administered in combination:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[2] The antitumor agent according to [1], wherein thepyrazolo[3,4-d]pyrimidine compound is a compound, wherein, in theformula (I),X is a 4- to 8-membered nitrogen-containing saturated heterocyclic groupoptionally having a halogen atom or a C1-C6 alkyl group as asubstituent;Y is —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a halogen atom, a cyano group, a C2-C6 alkenylgroup, an optionally halogen atom-substituted C1-C6 alkoxy group, aC1-C6 alkyl group, an optionally C1-C6 alkyl group-substituted aminogroup, a C3-C7 cycloalkyl group, or a monocyclic 4- to 6-memberedunsaturated heterocyclic group having one oxygen atom, or Z₁ and Z₂, orZ₃ and Z₄ optionally form, together with the respective carbon atomsbonded thereto, a benzene ring or a 5- to 7-membered saturated orunsaturated heterocyclic ring;W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group;R₂ and R₃ are the same as or different from each other and each are ahydrogen atom, a C1-C6 alkoxy group, a C1-C6 alkyl group, or a C6-C14aromatic hydrocarbon group, or R₂ and R₃ optionally form, together withthe nitrogen atom bonded thereto, an optionally hydroxylgroup-substituted 4- to 8-membered nitrogen-containing saturatedheterocyclic group; andR₄, R₅, and R₆ are the same as or different from each other and each area hydrogen atom or an optionally di(C1-C6 alkyl)amino group-substitutedC1-C6 alkyl group.[3] The antitumor agent according to [1] or [2], wherein thepyrazolo[3,4-d]pyrimidine compound is a compound, wherein, in theformula (I),X is a pyrrolidinyl group, a methylpyrrolidinyl group, a piperidinylgroup, or a fluoropiperidinyl group;

Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group, a fluoromethoxy group, adifluoromethoxy group, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring;

W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group; andR₂ and R₃ are the same as or different from each other and each are amethoxy group, a methyl group, or a phenyl group, or R₂ and R₃optionally form, together with the nitrogen atom bonded thereto, ahydroxyazetidinyl group, a pyrrolidinyl group, or a piperidinyl group.[4] The antitumor agent according to any of [1] to [3], wherein thepyrazolo[3,4-d]pyrimidine compound is a compound selected from the groupconsisting of the following (1) to (20):

-   (1)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-bromo-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (2)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (3)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (4)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (5)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (6)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (7)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (8)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (9)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (10)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-5-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (11)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (12)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (13)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (14)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (15)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-(difluoromethoxy)-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (16)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-(fluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (17)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-bromo-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (18)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (19)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,    and-   (20)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2,5-dichloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.    [5] The antitumor agent according to any of [1] to [4], wherein the    other antitumor agent(s) is at least one selected from the group    consisting of an antimetabolite, a molecular targeting drug, a    platinum-based drug, and an alkaloid-based drug.    [6] The antitumor agent according to any of [1] to [5], wherein the    other antitumor agent(s) is at least one selected from the group    consisting of 5-fluorouracil (5-FU), 5-fluoro-2′-deoxyuridine    (FdUrd), trifluridine, gemcitabine, capecitabine, trastuzumab,    trastuzumab emtansine, AZD8055, everolimus, dactolisib, buparlisib,    taselisib, palbociclib, cisplatin, and paclitaxel.    [7] An agent enhancing an antitumor effect of other antitumor    agent(s), the agent comprising a pyrazolo[3,4-d]pyrimidine compound    of the following formula (I) or a salt thereof as an active    ingredient:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[8] An antitumor agent for treating a cancer patient dosed with otherantitumor agent(s), the antitumor agent comprising apyrazolo[3,4-d]pyrimidine compound or a salt thereof, wherein

the pyrazolo[3,4-d]pyrimidine compound is a compound of the followingformula (I):

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R_(G));Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[9] An antitumor agent comprising a pyrazolo[3,4-d]pyrimidine compoundor a salt thereof and other antitumor agent(s) in combination, wherein

the pyrazolo[3, 4-d]pyrimidine compound is a compound of the followingformula (I):

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[10] A pyrazolo[3,4-d]pyrimidine compound of the following formula (I)or a salt thereof for use in the treatment of tumor by combinedadministration with other antitumor agent(s):

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[11] The pyrazolo[3,4-d]pyrimidine compound according to [10] or a saltthereof, wherein the pyrazolo[3,4-d]pyrimidine compound is a compound ofthe formula (I) whereinX is a 4- to 8-membered nitrogen-containing saturated heterocyclic groupoptionally having a halogen atom or a C1-C6 alkyl group as asubstituent;Y is —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a halogen atom, a cyano group, a C2-C6 alkenylgroup, an optionally halogen atom-substituted C1-C6 alkoxy group, aC1-C6 alkyl group, an optionally C1-C6 alkyl group-substituted aminogroup, a C3-C7 cycloalkyl group, or a monocyclic 4- to 6-memberedunsaturated heterocyclic group having one oxygen atom, or Z₁ and Z₂, orZ₃ and Z₄ optionally form, together with the respective carbon atomsbonded thereto, a benzene ring or a 5- to 7-membered saturated orunsaturated heterocyclic ring;W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group;R₂ and R₃ are the same as or different from each other and each are ahydrogen atom, a C1-C6 alkoxy group, a C1-C6 alkyl group, or a C6-C14aromatic hydrocarbon group, or R₂ and R₃ optionally form, together withthe nitrogen atom bonded thereto, an optionally hydroxylgroup-substituted 4- to 8-membered nitrogen-containing saturatedheterocyclic group; andR₄, R₅, and R₆ are the same as or different from each other and each area hydrogen atom or an optionally di(C1-C6 alkyl)amino group-substitutedC1-C6 alkyl group.[12] The pyrazolo[3,4-d]pyrimidine compound according to [10] or [11] ora salt thereof, wherein the pyrazolo[3,4-d]pyrimidine compound is acompound of the formula (I) whereinX is a pyrrolidinyl group, a methylpyrrolidinyl group, a piperidinylgroup, or a fluoropiperidinyl group;

Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group, a fluoromethoxy group, adifluoromethoxy group, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring;

W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group; andR₂ and R₃ are the same as or different from each other and each are amethoxy group, a methyl group, or a phenyl group, or R₂ and R₃optionally form, together with the nitrogen atom bonded thereto, ahydroxyazetidinyl group, a pyrrolidinyl group, or a piperidinyl group.[13] The pyrazolo[3,4-d]pyrimidine compound according to any of [10] to[12] or a salt thereof, wherein the pyrazolo[3,4-d]pyrimidine compoundis a compound selected from the group consisting of the following (1) to(20):

-   (1)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-bromo-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (2)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (3)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (4)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (5)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (6)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (7)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (8)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (9)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (10)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-5-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (11)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (12)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (13)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (14)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (15)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-(difluoromethoxy)-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (16)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-(fluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (17)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-bromo-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (18)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (19)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,    and-   (20)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2,5-dichloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.    [14] The pyrazolo[3,4-d]pyrimidine compound according to any of [10]    to [13] or a salt thereof, wherein the other antitumor agent(s) is    at least one selected from the group consisting of an    antimetabolite, a molecular targeting drug, a platinum-based drug,    and an alkaloid-based drug.    [15] The pyrazolo[3,4-d]pyrimidine compound according to any of [10]    to [14] or a salt thereof, wherein the other antitumor agent(s) is    at least one selected from the group consisting of 5-fluorouracil    (5-FU), 5-fluoro-2′-deoxyuridine (FdUrd), trifluridine, gemcitabine,    capecitabine, trastuzumab, trastuzumab emtansine, AZD8055,    everolimus, dactolisib, buparlisib, taselisib, palbociclib,    cisplatin, and paclitaxel.    [16] A pyrazolo[3,4-d]pyrimidine compound of the following    formula (I) or a salt thereof for use in the enhancement of an    antitumor effect of other antitumor agent(s):

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[17] A pyrazolo[3,4-d]pyrimidine compound of the following formula (I)or a salt thereof for use in the treatment of tumor in a cancer patientdosed with other antitumor agent(s):

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[18] A combination of a pyrazolo[3,4-d]pyrimidine compound of thefollowing formula (I) or a salt thereof and other antitumor agent(s) foruse in the treatment of tumor

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[19] Use of a pyrazolo[3,4-d]pyrimidine compound of the followingformula (I) or a salt thereof for producing an antitumor agent forcombined administration with other antitumor agent(s):

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[20] The use according to [19], wherein the pyrazolo[3,4-d]pyrimidinecompound is a compound of the formula (I) whereinX is a 4- to 8-membered nitrogen-containing saturated heterocyclic groupoptionally having a halogen atom or a C1-C6 alkyl group as asubstituent;Y is —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a halogen atom, a cyano group, a C2-C6 alkenylgroup, an optionally halogen atom-substituted C1-C6 alkoxy group, aC1-C6 alkyl group, an optionally C1-C6 alkyl group-substituted aminogroup, a C3-C7 cycloalkyl group, or a monocyclic 4- to 6-memberedunsaturated heterocyclic group having one oxygen atom, or Z₁ and Z₂, orZ₃ and Z₄ optionally form, together with the respective carbon atomsbonded thereto, a benzene ring or a 5- to 7-membered saturated orunsaturated heterocyclic ring;W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group;R₂ and R₃ are the same as or different from each other and each are ahydrogen atom, a C1-C6 alkoxy group, a C1-C6 alkyl group, or a C6-C14aromatic hydrocarbon group, or R₂ and R₃ optionally form, together withthe nitrogen atom bonded thereto, an optionally hydroxylgroup-substituted 4- to 8-membered nitrogen-containing saturatedheterocyclic group; and R₄, R₅, and R₆ are the same as or different fromeach other and each are a hydrogen atom or an optionally di(C1-C6alkyl)amino group-substituted C1-C6 alkyl group.[21] The use according to [19] or [20], wherein thepyrazolo[3,4-d]pyrimidine compound is a compound of the formula (I)whereinX is a pyrrolidinyl group, a methylpyrrolidinyl group, a piperidinylgroup, or a fluoropiperidinyl group;

Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group, a fluoromethoxy group, adifluoromethoxy group, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring;

W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group; andR₂ and R₃ are the same as or different from each other and each are amethoxy group, a methyl group, or a phenyl group, or R₂ and R₃optionally form, together with the nitrogen atom bonded thereto, ahydroxyazetidinyl group, a pyrrolidinyl group, or a piperidinyl group.[22] The use according to any of [19] to [21], wherein thepyrazolo[3,4-d]pyrimidine compound is a compound selected from the groupconsisting of the following (1) to (20):

-   (1)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-bromo-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (2)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (3)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (4)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (5)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (6)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (7)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (8)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (9)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxcethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (10)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-5-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (11)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (12)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (13)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (14)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (15)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-(difluoromethoxy)-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (16)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-(fluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (17)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-bromo-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (18)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (19)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,    and-   (20)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2,5-dichloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.    [23] The use according to any of [19] to [22], wherein the other    antitumor agent(s) is at least one selected from the group    consisting of an antimetabolite, a molecular targeting drug, a    platinum-based drug, and an alkaloid-based drug.    [24] The use according to any one of [19] to [23], wherein the other    antitumor agent(s) is at least one selected from the group    consisting of 5-fluorouracil (5-FU), 5-fluoro-2′-deoxyuridine    (FdUrd), trifluridine, gemcitabine, capecitabine, trastuzumab,    trastuzumab emtansine, AZD8055, everolimus, dactolisib, buparlisib,    taselisib, palbociclib, cisplatin, and paclitaxel.    [25] Use of a pyrazolo[3,4-d]pyrimidine compound of the following    formula (I) or a salt thereof for producing an agent enhancing an    antitumor effect of other antitumor agent(s):

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[26] Use of a pyrazolo[3,4-d]pyrimidine compound of the followingformula (I) or a salt thereof for producing an antitumor agent fortreating a cancer patient dosed with other antitumor agent(s):

wherein x represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[27] Use of a pyrazolo[3,4-d]pyrimidine compound of the followingformula (I) or a salt thereof for producing an antitumor agentcomprising other antitumor agent(s) in combination therewith:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[28] A method for treating tumor, comprising performing the combinedadministration of a pyrazolo[3,4-d]pyrimidine compound of the followingformula (I) or a salt thereof and other antitumor agent(s) to a subjectin need thereof:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₂ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[29] The treatment method according to [28], wherein thepyrazolo[3,4-d]pyrimidine compound is a compound of the formula (I)whereinX is a 4- to 8-membered nitrogen-containing saturated heterocyclic groupoptionally having a halogen atom or a C1-C6 alkyl group as asubstituent;Y is —C(R₄)=C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a halogen atom, a cyano group, a C2-C6 alkenylgroup, an optionally halogen atom-substituted C1-C6 alkoxy group, aC1-C6 alkyl group, an optionally C1-C6 alkyl group-substituted aminogroup, a C3-C7-cycloalkyl group, or a monocyclic 4- to 6-memberedunsaturated heterocyclic group having one oxygen atom, or Z_(x) and Z₂,or Z₃ and Z₄ optionally form, together with the respective carbon atomsbonded thereto, a benzene ring or a 5- to 7-membered saturated orunsaturated heterocyclic ring;W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group;R₂ and R₃ are the same as or different from each other and each are ahydrogen atom, a C1-C6 alkoxy group, a C1-C6 alkyl group, or a C6-C14aromatic hydrocarbon group, or R₂ and R₃ optionally form, together withthe nitrogen atom bonded thereto, an optionally hydroxylgroup-substituted 4- to 8-membered nitrogen-containing saturatedheterocyclic group; andR₄, R₅, and R₆ are the same as or different from each other and each area hydrogen atom or an optionally di(C1-C6 alkyl)amino group-substitutedC1-C6 alkyl group.[30] The treatment method according to [28] or [29], wherein thepyrazolo[3,4-d]pyrimidine compound is a compound of the formula (I)whereinX is a pyrrolidinyl group, a methylpyrrolidinyl group, a piperidinylgroup, or a fluoropiperidinyl group;

Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group, a fluoromethoxy group, adifluoromethoxy group, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring;

W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group; andR₂ and R₃ are the same as or different from each other and each are amethoxy group, a methyl group, or a phenyl group, or R₂ and R₃optionally form, together with the nitrogen atom bonded thereto, ahydroxyazetidinyl group, a pyrrolidinyl group, or a piperidinyl group.[31] The treatment method according to any of [28] to [30], wherein thepyrazolo[3,4-d]pyrimidine compound is a compound selected from the groupconsisting of the following (1) to (20):

-   (1)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-bromo-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (2)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (3)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (4)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (5)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (6)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (7)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (8)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3    carboxamide,-   (9)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (10)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-5-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (11)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (12)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (13)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (14)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (15)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-(difluoromethoxy)-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (16)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-(fluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (17)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-bromo-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (18)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (19)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,    and-   (20)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2,5-dichloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.    [32] The treatment method according to any of [28] to [31], wherein    the other antitumor agent(s) is at least one selected from the group    consisting of an antimetabolite, a molecular targeting drug, a    platinum-based drug, and an alkaloid-based drug.    [33] The treatment method according to any of [28] to [32], wherein    the other antitumor agent(s) is at least one selected from the group    consisting of 5-fluorouracil (5-FU), 5-fluoro-2′-deoxyuridine    (FdUrd), trifluridine, gemcitabine, capecitabine, trastuzumab,    trastuzumab emtansine, AZD8055, everolimus, dactolisib, buparlisib,    taselisib, palbociclib, cisplatin, and paclitaxel.    [34] A method for enhancing an antitumor effect of other antitumor    agent(s), comprising administering a pyrazolo[3,4-d]pyrimidine    compound of the following formula (I) or a salt thereof to a subject    in need thereof:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆));Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[35] A method for treating tumor in a cancer patient dosed with otherantitumor agent(s), comprising administering a pyrazolo[3,4-d]pyrimidinecompound of the following formula (I) or a salt thereof to a subject inneed thereof:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;

Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R₆ are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.[36] A method for treating tumor, comprising administering a combinationof pyrazolo[3,4-d]pyrimidine compound of the following formula (I) or asalt thereof and other antitumor agent(s) to a subject in need thereof:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group;Y represents —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring;W represents —CH₂—, an oxygen atom, or —NH—;n represents an integer of from 0 to 2;R₁ represents an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; andR₄, R₅, and R_(E) are the same as or different from each other and eachrepresent a hydrogen atom or an optionally substituted C1-C6 alkylgroup.

Effects of the Invention

The present invention enables cancer treatment which produces anexcellent antitumor effect while preventing adverse effects.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing the effect of combined use of trastuzumaband Compound 1.

FIG. 2 is a diagram showing the effect of combined use of trastuzumabemtansine and Compound 1.

FIG. 3 is a diagram showing the effect of combined use of capecitabine(359 mg/kg/day) and Compound 1.

FIG. 4 is a diagram showing the effect of combined use of capecitabine(809 mg/kg/day) and Compound 1.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the HER2 inhibitor which brings about anexcellent synergistic effect with other antitumor agent(s) is apyrazolo[3,4-d]pyrimidine compound of the following formula (I) or asalt thereof:

wherein X, Y, Z₁ to Z₄, W, n, and R₁ to R₃ are as defined above.

In the present specification, examples of the “substituent” include ahalogen atom, a hydroxyl group, a cyano group, a nitro group, an alkylgroup, a halogenoalkyl group, a cycloalkyl group, a cycloalkyl-alkylgroup, an aralkyl group, an alkenyl group, an alkynyl group, an alkoxygroup, a halogenoalkoxy group, a cycloalkoxy group, a cycloalkyl-alkoxygroup, an aralkyloxy group, an alkylthio group, a cycloalkyl-alkylthiogroup, an amino group, a mono- or di-alkylamino group, acycloalkyl-alkylamino group, an acyl group, an acyloxy group, an oxogroup, a carboxyl group, an alkoxycarbonyl group, an aralkyloxycarbonylgroup, a carbamoyl group, a saturated or unsaturated heterocyclic group,an aromatic hydrocarbon group, and a saturated heterocyclyloxy group.When the substituent is present, the number thereof is typically 1, 2,or 3.

In the present specification, examples of the “halogen atom” include afluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

In the present specification, the “alkyl group” may be linear orbranched. Examples thereof include C1-C6 alkyl groups such as a methylgroup, an ethyl group, a n-propyl group, an isopropyl group, a n-butylgroup, an isobutyl group, a sec-butyl group, a tert-butyl group, an-pentyl group, an isopentyl group, and a hexyl group.

In the present specification, the “halogenoalkyl group” is a linear orbranched alkyl group having 1 to 6 carbon atoms and having 1 to 13halogen atoms (halogeno-C1-C6 alkyl group). Examples thereof includehalogeno-C1-C6 alkyl groups such as a fluoromethyl group, adifluoromethyl group, a trifluoromethyl group, a trichloromethyl group,a fluoroethyl group, a 1,1,1-trifluoroethyl group, a monofluoro-n-propylgroup, a perfluoro-n-propyl group, and a perfluoroisopropyl group andpreferably include halogeno-C1-C4 alkyl groups.

In the present specification, specific examples of the “cycloalkylgroup” include C3-C7 cycloalkyl groups such as a cyclopropyl group, acyclobutyl group, a cyclopentyl group, a cyclohexyl group, and acycloheptyl group.

In the present specification, examples of the “cycloalkyl-alkyl group”include C3-C7 cycloalkyl-substituted C1-C4 alkyl groups such as acyclopropylmethyl group, a cyclobutylmethyl group, a cyclopentylmethylgroup, a cyclohexylmethyl group, and a cycloheptylmethyl group.

In the present specification, examples of the “aralkyl group” includeC7-C13 aralkyl groups such as a benzyl group, a phenethyl group, anaphthylmethyl group, and a fluorenylmethyl group.

In the present specification, the “alkenyl group” may be linear,branched, or cyclic and means an unsaturated hydrocarbon group having atleast one double bond. Examples thereof include C2-C6 alkenyl groupssuch as a vinyl group, an allyl group, a 1-propenyl group, a2-methyl-2-propenyl group, an isopropenyl group, a 1-, 2-, or 3-butenylgroup, a 2-, 3-, or 4-pentenyl group, a 2-methyl-2-butenyl group, a3-methyl-2-butenyl group, a 5-hexenyl group, a 1-cyclopentenyl group, a1-cyclohexenyl group, and a 3-methyl-3-butenyl group.

In the present specification, the “alkynyl group” may be linear,branched, or cyclic and means an unsaturated hydrocarbon group having atleast one triple bond. Examples thereof include C2-C6 alkynyl groupssuch as an ethynyl group, a 1- or 2-propynyl group, a 1-, 2-, or3-butynyl group, and a 1-methyl-2-propynyl group.

In the present specification, the “alkoxy group” may be linear orbranched. Examples thereof include C1-C6 alkoxy groups such as a methoxygroup, an ethoxy group, a n-propoxy group, an isopropoxy group, an-butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxygroup, a pentyloxy group, an isopentyloxy group, and a hexyloxy group.

In the present specification, the “halogenoalkoxy group” is a linear orbranched alkoxy group having 1 to 6 carbon atoms and having 1 to 13halogen atoms (halogeno-C1-C6 alkoxy group). Examples thereof includehalogeno-C1-C6 alkoxy groups such as a fluoromethoxy group, adifluoromethoxy group, a trifluoromethoxy group, a trichloromethoxygroup, a fluoroethoxy group, a 1,1,1-trifluoroethoxy group, amonofluoro-n-propoxy group, a perfluoro-n-propoxy group, and aperfluoro-isopropoxy group and preferably include halogeno-C1-C4 alkoxygroups.

In the present specification, examples of the “cycloalkoxy group”include C3-C7 cycloalkoxy groups such as a cyclopropoxy group, acyclobutoxy group, a cyclopentyloxy group, a cyclohexyloxy group, and acycloheptyloxy group.

In the present specification, examples of the “cycloalkyl-alkoxy group”include C3-C7 cycloalkyl-substituted C1-C4 alkoxy groups such as acyclopropylmethoxy group, a cyclobutylmethoxy group, acyclopentylmethoxy group, a cyclohexylmethoxy group, and acycloheptylmethoxy group.

In the present specification, examples of the “aralkyloxy group” includeC7-C13 aralkyloxy groups such as a benzyloxy group, a phenethyloxygroup, a naphthylmethyloxy group, and a fluorenylmethyloxy group.

In the present specification, the “alkylthio group” may be linear orbranched. Examples thereof include C1-C6 alkylthio groups such as amethylthio group, an ethylthio group, a n-propylthio group, anisopropylthio group, a n-butylthio group, an isobutylthio group, atert-butylthio group, a n-pentylthio group, an isopentylthio group, anda hexylthio group.

In the present specification, examples of the “cycloalkyl-alkylthiogroup” include C3-C7 cycloalkyl-substituted C1-C4 alkylthio groups suchas a cyclopropylmethylthio group, a cyclobutylmethylthio group, acyclopentylmethylthio group, a cyclohexylmethylthio group, and acycloheptylmethylthio group.

In the present specification, examples of the “monoalkylamino group”include linear or branched C1-C6 alkyl group-monosubstituted aminogroups such as a methylamino group, an ethylamino group, a n-propylaminogroup, an isopropylamino group, a n-butylamino group, an isobutylaminogroup, a tert-butylamino group, a n-pentylamino group, an isopentylaminogroup, and a hexylamino group.

In the present specification, examples of the “dialkylamino group”include linear or branched C1-C6 alkyl group-disubstituted amino groupssuch as a dimethylamino group, a diethylamino group, a di(n-propyl)aminogroup, a diisopropylamino group, a di(n-butyl)amino group, adiisobutylamino group, a di(tert-butyl)amino group, a di(n-pentyl)aminogroup, a diisopentylamino group, a dihexylamino group, amethylethylamino group, and a methylisopropylamino group.

In the present specification, examples of the “cycloalkyl-alkylaminogroup” include C3-C7 cycloalkyl-substituted C1-C4 alkylamino groups suchas a cyclopropylmethylamino group, a cyclobutylmethylamino group, acyclopentylmethylamino group, a cyclohexylmethylamino group, and acycloheptylmethylamino group.

In the present specification, the “acyl group” means an alkylcarbonylgroup or an arylcarbonyl group.

In the present specification, examples of the “alkylcarbonyl group”include linear or branched (C1-C6 alkyl)carbonyl groups such as amethylcarbonyl group, an ethylcarbonyl group, a n-propylcarbonyl group,an isopropylcarbonyl group, a n-butylcarbonyl group, an isobutylcarbonylgroup, a tert-butylcarbonyl group, a n-pentylcarbonyl group, anisopentylcarbonyl group, and a hexylcarbonyl group.

In the present specification, examples of the “arylcarbonyl group”include (C6-C13 aryl)carbonyl groups such as a phenylcarbonyl group, anaphthylcarbonyl group, a fluorenylcarbonyl group, an anthrylcarbonylgroup, a biphenylylcarbonyl group, a tetrahydronaphthylcarbonyl group, achromanylcarbonyl group, a 2,3-dihydro-1,4-dioxanaphthalenylcarbonylgroup, an indanylcarbonyl group, and a phenanthrylcarbonyl group.

In the present specification, the “acyloxy group” means analkylcarbonyloxy group or an arylcarbonyloxy group.

In the present specification, examples of the “alkylcarbonyloxy group”include linear or branched (C1-C6 alkyl)carbonyloxy groups such as amethylcarbonyloxy group, an ethylcarbonyloxy group, an-propylcarbonyloxy group, an isopropylcarbonyloxy group, an-butylcarbonyloxy group, an isobutylcarbonyloxy group, atert-butylcarbonyloxy group, a n-pentylcarbonyloxy group, anisopentylcarbonyloxy group, and a hexylcarbonyloxy group.

In the present specification, examples of the “arylcarbonyloxy group”include (C6-C13 aryl)carbonyloxy groups such as a phenylcarbonyloxygroup, a naphthylcarbonyloxy group, a fluorenylcarbonyloxy group, ananthrylcarbonyloxy group, a biphenylylcarbonyloxy group, atetrahydronaphthylcarbonyloxy group, a chromanylcarbonyloxy group, a2,3-dihydro-1,4-dioxanaphthalenylcarbonyloxy group, anindanylcarbonyloxy group, and a phenanthrylcarbonyloxy group.

In the present specification, the “alkoxycarbonyl group” may be linearor branched. Examples thereof include (C1-C6 alkoxy)carbonyl groups suchas a methoxycarbonyl group, an ethoxycarbonyl group, a propoxycarbonylgroup, an isopropoxycarbonyl group, a butoxycarbonyl group, anisobutoxycarbonyl group, a tert-butoxycarbonyl group, apentyloxycarbonyl group, an isopentyloxycarbonyl group, and ahexyloxycarbonyl group.

In the present specification, examples of the “aralkyloxycarbonyl group”include (C7-C13 aralkyl)oxycarbonyl groups such as a benzyloxycarbonylgroup, a phenethyloxycarbonyl group, a naphthylmethyloxycarbonyl group,and a fluorenylmethyloxycarbonyl group.

In the present specification, the “saturated heterocyclic group” is asaturated heterocyclic group having a heteroatom selected from the groupconsisting of a nitrogen atom, an oxygen atom, and a sulfur atom.Specific examples thereof include a morpholino group, a pyrrolidinylgroup, a piperidinyl group, a piperazinyl group, a4-methyl-1-piperazinyl group, a tetrahydrofuranyl group, atetrahydropyranyl group, a tetrahydrothiophenyl group, a thiazolidinylgroup, and an oxazolidinyl group.

In the present specification, the “unsaturated heterocyclic group” is amonocyclic or polycyclic fully unsaturated or partially unsaturatedheterocyclic group having a heteroatom selected from the groupconsisting of a nitrogen atom, an oxygen atom, and a sulfur atom.Specific examples thereof include an imidazolyl group, a thienyl group,a furyl group, a pyrrolyl group, an oxazolyl group, an isoxazolyl group,a thiazolyl group, an isothiazolyl group, a thiadiazolyl group, apyrazolyl group, a triazolyl group, a tetrazolyl group, a pyridyl group,a pyrazyl group, a pyrimidinyl group, a pyridazinyl group, an indolylgroup, an isoindolyl group, an indazolyl group, a triazolopyridyl group,a benzimidazolyl group, a benzoxazolyl group, a benzothiazolyl group, abenzothienyl group, a benzofuranyl group, a purinyl group, a quinolylgroup, an isoquinolyl group, a quinazolinyl group, a quinoxalinyl group,a methylenedioxyphenyl group, an ethylenedioxyphenyl group, and adihydrobenzofuranyl group.

In the present specification, examples of the “aromatic hydrocarbongroup” include C6-C14 aromatic hydrocarbon groups such as a phenylgroup, a toluyl group, a xylyl group, a naphthyl group, an anthracenylgroup, a phenanthryl group, a fluorenyl group, and a tetrahydronaphthylgroup.

In the present specification, the “saturated heterocyclyloxy group” is asaturated heterocyclyloxy group having a heteroatom selected from thegroup consisting of a nitrogen atom, an oxygen atom, and a sulfur atom.Specific examples thereof include a morpholinyloxy group, a1-pyrrolidinyloxy group, a piperidinoxy group, a piperazinyloxy group, a4-methyl-1-piperazinyloxy group, a tetrahydrofuranyloxy group, atetrahydropyranyloxy group, a tetrahydrothiophenyloxy group, athiazolidinyloxy group, and an oxazolidinyloxy group.

The term “CA-CB” in the description of a group in the presentspecification refers to a group having A to B carbon atoms. For example,the “C1-C6 alkyl group” refers to an alkyl group having 1 to 6 carbonatoms, and the “C6-C14 aromatic hydrocarbon oxy group” refers to an oxygroup bonded to an aromatic hydrocarbon group having 6 to 14 carbonatoms. The term “A- to B-membered” means that the number of atomsconstituting a ring (the number of ring members) is A to B. For example,the “4- to 10-membered saturated heterocyclic group” means a saturatedheterocyclic group in which the number of ring members is 4 to 10.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I) of the presentinvention, X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group. In this context, the“4- to 10-membered nitrogen-containing saturated heterocyclic group” isa 4- to 10-membered saturated heterocyclic group containing at least onenitrogen atom in the ring and further containing 0 to 2 identical ordifferent heteroatoms selected from the group consisting of an oxygenatom and a sulfur atom in the ring. Examples thereof include anazetidinyl group, a pyrrolidinyl group, a piperidinyl group, apiperazinyl group, a morpholinyl group, an octahydroquinolinylene group,and an octahydroindolylene group.

The 4- to 10-membered nitrogen-containing saturated heterocyclic groupis preferably a 4- to 8-membered saturated heterocyclic group containingone nitrogen atom in the ring, more preferably a pyrrolidinyl group or apiperidinyl group, even more preferably a 1,3-pyrrolidinyl group or a1,3-piperidinyl group, further preferably a 1,3-piperidinyl group.

Examples of the “substituent” which may be added to the 4- to10-membered nitrogen-containing saturated heterocyclic group include thesubstituent as described above. The substituent is preferably a halogenatom, a hydroxyl group, a cyano group, a C1-C6 alkyl group, or an aminogroup, more preferably a halogen atom or a C1-C6 alkyl group, even morepreferably a fluorine atom or a methyl group, further preferably afluorine atom.

The optionally halogen atom- or C1-C6 alkyl group-substituted 4- to10-membered nitrogen-containing saturated heterocyclic group ispreferably an optionally halogen atom- or C1-C6 alkyl group-substitutedpyrrolidinyl group, or an optionally halogen atom- or C1-C6 alkylgroup-substituted piperidinyl group, more preferably an optionally C1-C6alkyl group-substituted pyrrolidinyl group, or an optionally fluorineatom-substituted piperidinyl group, even more preferably a pyrrolidinylgroup, a methylpyrrolidinyl group, a piperidinyl group, or afluoropiperidinyl group, further preferably a piperidinyl group or afluoropiperidinyl group.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I), X ispreferably an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group, more preferably a 4-to 10-membered nitrogen-containing saturated heterocyclic groupoptionally having a halogen atom, a hydroxyl group, a cyano group, aC1-C6 alkyl group, or an amino group as a substituent, even morepreferably a 4- to 8-membered nitrogen-containing saturated heterocyclicgroup optionally having a halogen atom or a C1-C6 alkyl group as asubstituent, further preferably a pyrrolidinyl group optionally having ahalogen atom or a C1-C6 alkyl group as a substituent, or a piperidinylgroup optionally having a halogen atom or a C1-C6 alkyl group as asubstituent, still further preferably a pyrrolidinyl group optionallyhaving a C1-C6 alkyl group as a substituent, or a piperidinyl groupoptionally having a halogen atom as a substituent, still furtherpreferably a pyrrolidinyl group optionally having a methyl group as asubstituent, or a piperidinyl group optionally having a fluorine atom asa substituent, still further preferably a piperidinyl group optionallyhaving a fluorine atom as a substituent, still further preferably apiperidinyl group.

The nitrogen atom in the 4- to 10-membered nitrogen-containing saturatedheterocyclic group represented by X is preferably bonded to the carbonylgroup of —COY in the formula (I).

In the pyrazolo[3,4-d]pyrimidine compound of formula (I) of the presentinvention, Y represents —C(R₄)═C(R₅)(R₆). R₄, R₅, and R will bementioned later.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I) of the presentinvention, Z₁, Z₂, Z₃, and Z₄ are the same as or different from eachother and each represent a hydrogen atom, a halogen atom, a cyano group,a C2-C6 alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring.

The “C2-C6 alkenyl group” represented by Z₁, Z₂, Z₃, or Z₄ is preferablya vinyl group, a propenyl group, a butenyl group, a pentenyl group, ahexenyl group, a cyclopropenyl group, a cyclobutenyl group, acyclopentenyl group, or a cyclohexenyl group, more preferably a vinylgroup, a propenyl group, a butenyl group, a cyclopropenyl group, or acyclobutenyl group, even more preferably a vinyl group, a 1-propenylgroup, a 2-propenyl group, or an isopropenyl group, further preferably avinyl group.

The “C1-C6 alkoxy group” in the “optionally substituted C1-C6 alkoxygroup” represented by Z₁, Z₂, Z₃, or Z₄ is preferably a methoxy group,an ethoxy group, a propoxy group, a butoxy group, a pentyloxy group, ora hexyloxy group, more preferably a methoxy group, an ethoxy group, apropoxy group, or a butoxy group, even more preferably a methoxy group,an ethoxy group, a n-propoxy group, an isopropoxy group, a n-butoxygroup, a sec-butoxy group, or a tert-butoxy group, further preferably amethoxy group.

Examples of the “substituent” for the “optionally substituted C1-C6alkoxy group” represented by Z₂, Z₂, Z₃, or Z₄ include the substituentas described above. The substituent is preferably a halogen atom, morepreferably a fluorine atom.

The “optionally substituted C1-C6 alkoxy group” represented by Z₁, Z₂,Z₃, or Z₄ is preferably an optionally halogen atom-substituted C1-C6alkoxy group, more preferably an optionally fluorine atom-substitutedC1-C6 alkoxy group, even more preferably an optionally fluorineatom-substituted methoxy group, an optionally fluorine atom-substitutedethoxy group, an optionally fluorine atom-substituted propoxy group, anoptionally fluorine atom-substituted butoxy group, an optionallyfluorine atom-substituted pentyloxy group, or an optionally fluorineatom-substituted hexyloxy group, further preferably an optionallyfluorine atom-substituted methoxy group, an optionally fluorineatom-substituted ethoxy group, an optionally fluorine atom-substitutedpropoxy group, or an optionally fluorine atom-substituted butoxy group,still further preferably an optionally fluorine atom-substituted methoxygroup, an optionally fluorine atom-substituted ethoxy group, anoptionally fluorine atom-substituted n-propoxy group, an optionallyfluorine atom-substituted isopropoxy group, an optionally fluorineatom-substituted n-butoxy group, an optionally fluorine atom-substitutedsec-butoxy group, or an optionally fluorine atom-substituted tert-butoxygroup, still further preferably a methoxy group optionally substitutedby 1 or 2 fluorine atoms, an ethoxy group optionally substituted by 1 or2 fluorine atoms, a n-propoxy group optionally substituted by 1 or 2fluorine atoms, an isopropoxy group optionally substituted by 1 or 2fluorine atoms, a n-butoxy group optionally substituted by 1 or 2fluorine atoms, a sec-butoxy group optionally substituted by 1 or 2fluorine atoms, or a tert-butoxy group optionally substituted by 1 or 2fluorine atoms, particularly preferably a methoxy group, a fluoromethoxygroup, or a difluoromethoxy group.

The “C1-C6 alkyl group” in the “optionally substituted C1-C6 alkylgroup” represented by Z₁, Z₂, Z₃, or Z₄ is preferably a methyl group, anethyl group, a propyl group, a butyl group, a pentyl group, or a hexylgroup, more preferably a methyl group, an ethyl group, a n-propyl group,an isopropyl group, a n-butyl group, an isobutyl group, a sec-butylgroup, or a tert-butyl group, further preferably a methyl group or anethyl group.

Examples of the “substituent” for the “optionally substituted C1-C6alkyl group” represented by Z₁, Z₂, Z₃, or Z₄ include the substituent asdescribed above.

The “optionally substituted C1-C6 alkyl group” represented by Z₁, Z₂,Z₃, or Z₄ is preferably an unsubstituted C1-C6 alkyl group, morepreferably a methyl group, an ethyl group, a propyl group, a butylgroup, a pentyl group, or a hexyl group, further preferably a methylgroup, an ethyl group, a n-propyl group, an isopropyl group, a n-butylgroup, an isobutyl group, a sec-butyl group, or a tert-butyl group,particularly preferably a methyl group or an ethyl group.

Examples of the “substituent” for the “optionally substituted aminogroup” represented by Z₁, Z₂, Z₃, or Z₄ include the substituent asdescribed above. The substituent is preferably an alkyl group, morepreferably a C1-C6 alkyl group, even more preferably a methyl group, anethyl group, a propyl group, a butyl group, a pentyl group, or a hexylgroup, further preferably a methyl group, an ethyl group, a n-propylgroup, an isopropyl group, a n-butyl group, an isobutyl group, asec-butyl group, or a tert-butyl group, particularly preferably a methylgroup.

The optionally substituted amino group represented by Z₁, Z₂, Z₃, or Z₄is preferably a mono- or di-alkylamino group, more preferably a mono- ordi(C1-C6 alkyl)amino group, even more preferably a di(C1-C6 alkyl)aminogroup, further preferably a di(C1-C4 alkyl)amino group, still furtherpreferably a dimethylamino group.

The “C3-C7 cycloalkyl group” in the “optionally substituted C3-C7cycloalkyl group” represented by Z₁, Z₂, Z₃, or Z₄ is preferably acyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexylgroup, or a cycloheptyl group, more preferably a cyclopropyl group, acyclobutyl group, a cyclopentyl group, or a cyclohexyl group, furtherpreferably a cyclopropyl group.

Examples of the “substituent” for the “optionally substituted C3-C7cycloalkyl group” represented by Z₁, Z₂, Z₃, or Z₄ include thesubstituent as described above.

The “optionally substituted C3-C7 cycloalkyl group” represented by Z₁,Z₂, Z₃, or Z₄ is preferably an unsubstituted C3-C7 cycloalkyl group,more preferably a cyclopropyl group, a cyclobutyl group, a cyclopentylgroup, a cyclohexyl group, or a cycloheptyl group, further preferably acyclopropyl group, a cyclobutyl group, a cyclopentyl group, or acyclohexyl group, particularly preferably a cyclopropyl group.

The “C6-C14 aromatic hydrocarbon group” represented by Z₁, Z₂, Z₃, or Z₄is preferably a phenyl group or a naphthyl group, more preferably aphenyl group.

The “unsaturated heterocyclic group” represented by Z₁, Z₂, Z₃, or Z₄ isa monocyclic or polycyclic fully unsaturated or partially unsaturatedheterocyclic group having a heteroatom selected from the groupconsisting of a nitrogen atom, an oxygen atom, and a sulfur atom.Examples thereof include an imidazolyl group, a thienyl group, a furylgroup, a pyrrolyl group, an oxazolyl group, an isoxazolyl group, athiazolyl group, an isothiazolyl group, a thiadiazolyl group, apyrazolyl group, a triazolyl group, a tetrazolyl group, a pyridyl group,a pyrazyl group, a pyrimidinyl group, a pyridazinyl group, an indolylgroup, an isoindolyl group, an indazolyl group, a triazolopyridyl group,a benzimidazolyl group, a benzoxazolyl group, a benzothiazolyl group, abenzothienyl group, a benzofuranyl group, a purinyl group, a quinolylgroup, an isoquinolyl group, a quinazolinyl group, a quinoxalinyl group,a methylenedioxyphenyl group, an ethylenedioxyphenyl group, and adihydrobenzofuranyl group. The unsaturated heterocyclic group ispreferably a monocyclic or bicyclic 4- to 14-membered unsaturatedheterocyclic group having one heteroatom selected from the groupconsisting of a nitrogen atom, an oxygen atom, and a sulfur atom, morepreferably a monocyclic 4- to 6-membered fully unsaturated heterocyclicgroup having one heteroatom selected from the group consisting of anitrogen atom, an oxygen atom, and a sulfur atom, even more preferably amonocyclic 4- to 6-membered fully unsaturated heterocyclic group havingone oxygen atom, further preferably a furyl group.

The “5- to 7-membered saturated or unsaturated heterocyclic ring” formedby Z₁ and Z₂, or Z₃ and Z₄, together with the carbon atoms bondedthereto, is a 5- to 7-membered saturated, fully unsaturated, orpartially unsaturated heterocyclic ring having a heteroatom selectedfrom the group consisting of a nitrogen atom, an oxygen atom, and asulfur atom.

The number of the heteroatom in the ring is preferably from 0 to 2, morepreferably from 1 or 2. The heteroatom is preferably a nitrogen atomand/or an oxygen atom.

The 5- to 7-membered saturated heterocyclic ring or unsaturatedheterocyclic ring is preferably a pyrrole ring, a pyrazole ring, animidazole ring, a pyridine ring, a pyrazine ring, a pyrimidine ring, ora dioxolane ring, more preferably a pyridine ring or a dioxolane ring.

The ring formed by Z₁ and Z₂, or Z₃ and Z₄, together with the respectivecarbon atoms bonded thereto, is also preferably a benzene ring.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I), Z₁, Z₂, Z₃,and Z₄ are preferably the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, an optionally substitutedamino group, an optionally substituted C3-C7 cycloalkyl group, a C6-C14aromatic hydrocarbon group, or a monocyclic or bicyclic 4- to14-membered unsaturated heterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring or a 5- to 7-membered saturated or unsaturatedheterocyclic ring, more preferably are the same as or different fromeach other and each represent a hydrogen atom, a halogen atom, a cyanogroup, a C2-C6 alkenyl group, an optionally substituted C1-C6 alkoxygroup, an optionally substituted C1-C6 alkyl group, an optionallysubstituted amino group, an optionally substituted C3-C7 cycloalkylgroup, or a monocyclic or bicyclic 4- to 14-membered unsaturatedheterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring, even morepreferably are the same as or different from each other and eachrepresent a hydrogen atom, a halogen atom, a cyano group, a C2-C6alkenyl group, an optionally substituted C1-C6 alkoxy group, a C1-C6alkyl group, an optionally substituted amino group, a C3-C7 cycloalkylgroup, or a monocyclic 4- to 6-membered unsaturated heterocyclic grouphaving one heteroatom selected from the group consisting of a nitrogenatom, an oxygen atom, and a sulfur atom, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring or a 5- to 7-membered saturated or unsaturatedheterocyclic ring, further preferably are the same as or different fromeach other and each represent a hydrogen atom, a halogen atom, a cyanogroup, a C2-C6 alkenyl group, an optionally halogen atom-substitutedC1-C6 alkoxy group, a C1-C6 alkyl group, an optionally C1-C6 alkylgroup-substituted amino group, a C3-C7 cycloalkyl group, or a monocyclic4- to 6-membered unsaturated heterocyclic group having one heteroatomselected from the group consisting of a nitrogen atom, an oxygen atom,and a sulfur atom, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, togetherwith the respective carbon atoms bonded thereto, a benzene ring or a 5-to 7-membered saturated or unsaturated heterocyclic ring, still furtherpreferably are the same as or different from each other and eachrepresent a hydrogen atom, a fluorine atom, a chlorine atom, a bromineatom, a cyano group, a C2-C6 alkenyl group, an optionally fluorineatom-substituted C1-C6 alkoxy group, a C1-C6 alkyl group, a mono- ordi(C1-C6 alkyl)amino group, a C3-C7 cycloalkyl group, or a monocyclic 4-to 6-membered unsaturated heterocyclic group having one oxygen atom, orZ₁ and Z₂, or Z₃ and Z₄ optionally form, together with the respectivecarbon atoms bonded thereto, a benzene ring or a 5- to 7-memberedsaturated or unsaturated heterocyclic ring, still further preferably arethe same as or different from each other and each represent a hydrogenatom, a fluorine atom, a chlorine atom, a bromine atom, a cyano group, aC2-C6 alkenyl group, a C1-C6 alkoxy group optionally substituted by 1 or2 fluorine atoms, a C1-C6 alkyl group, a di(C1-C6 alkyl)amino group, aC3-C7 cycloalkyl group, or a monocyclic 4- to 6-membered unsaturatedheterocyclic group having one oxygen atom, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring or a 5- to 7-membered saturated or unsaturatedheterocyclic ring, still further preferably are the same as or differentfrom each other and each represent a hydrogen atom, a fluorine atom, achlorine atom, a bromine atom, a cyano group, a vinyl group, a methoxygroup, a fluoromethoxy group, a difluoromethoxy group, a methyl group,an ethyl group, a dimethylamino group, a cyclopropyl group, or a furylgroup, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, together with therespective carbon atoms bonded thereto, a benzene ring, a pyridine ring,or a dioxolane ring, still further preferably are the same as ordifferent from each other and each represent a hydrogen atom, a fluorineatom, a chlorine atom, a bromine atom, a methoxy group, a fluoromethoxygroup, a difluoromethoxy group, or a methyl group, or Z₁ and Z₂, or Z₃and Z₄ optionally form, together with the respective carbon atoms bondedthereto, a benzene ring.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I) of the presentinvention, W represents —CH₂—, an oxygen atom, or —NH—. W is preferably—CH₂— or an oxygen atom, more preferably —CH₂—.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I) of the presentinvention, n represents an integer of from 0 to 2. n is preferably 0 or1, more preferably 0.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I) of the presentinvention, R₁ represents an optionally substituted amino group. In thiscontext, examples of the “substituent” which may be added to the aminogroup include the substituent as described above.

The optionally substituted amino group represented by R₁ is preferablyan unsubstituted amino group.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I) of the presentinvention, R₂ and R₃ are the same as or different from each other andeach represent a hydrogen atom, an optionally substituted C1-C6 alkoxygroup, an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup.

The “C1-C6 alkoxy group” in the “optionally substituted C1-C6 alkoxygroup” represented by R₂ or R₃ is preferably a methoxy group, an ethoxygroup, a n-propoxy group, an isopropoxy group, a n-butoxy group, anisobutoxy group, a sec-butoxy group, a tert-butoxy group, a pentyloxygroup, or a hexyloxy group, more preferably a methoxy group, an ethoxygroup, a n-propoxy group, an isopropoxy group, a n-butoxy group, anisobutoxy group, a sec-butoxy group, or a tert-butoxy group, furtherpreferably a methoxy group.

Examples of the “substituent” for the optionally substituted C1-C6alkoxy group represented by R₂ or R₃ include the substituent asdescribed above.

The “optionally substituted C1-C6 alkoxy group” represented by R₂ or R₃is preferably an unsubstituted C1-C6 alkoxy group, more preferably amethoxy group, an ethoxy group, a n-propoxy group, an isopropoxy group,a n-butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxygroup, a pentyloxy group, or a hexyloxy group, further preferably amethoxy group, an ethoxy group, a n-propoxy group, an isopropoxy group,a n-butoxy group, an isobutoxy group, a sec-butoxy group, or atert-butoxy group, particularly preferably a methoxy group.

The “C1-C6 alkyl group” in the “optionally substituted C1-C6 alkylgroup” represented by R₂ or R₃ is preferably a methyl group, an ethylgroup, a n-propyl group, an isopropyl group, a n-butyl group, anisobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group,or a hexyl group, more preferably a methyl group, an ethyl group, an-propyl group, an isopropyl group, a n-butyl group, an isobutyl group,a sec-butyl group, or a tert-butyl group, further preferably a methylgroup.

Examples of the “substituent” for the “optionally substituted C1-C6alkyl group” represented by R₂ or R₃ include the substituent asdescribed above. The substituent is preferably a C1-C6 alkoxy group,preferably a methoxy group, an ethoxy group, a n-propoxy group, anisopropoxy group, a n-butoxy group, an isobutoxy group, a sec-butoxygroup, a tert-butoxy group, a pentyloxy group, or a hexyloxy group, morepreferably a methoxy group, an ethoxy group, a n-propoxy group, anisopropoxy group, a n-butoxy group, an isobutoxy group, a sec-butoxygroup, or a tert-butoxy group, further preferably a methoxy group or anethoxy group.

The “optionally substituted C1-C6 alkyl group” represented by R₂ or R₃is preferably an unsubstituted C1-C6 alkyl group or a C1-C6 alkoxygroup-substituted C1-C6 alkyl group, more preferably an unsubstitutedC1-C6 alkyl group, even more preferably a methyl group, an ethyl group,a n-propyl group, an isopropyl group, a n-butyl group, an isobutylgroup, a sec-butyl group, a tert-butyl group, a pentyl group, or a hexylgroup.

In the case of having a substituent, the number of the substituent isnot particularly limited. When the substituent is a C1-C6 alkoxy group,the number thereof is preferably 1.

The “C6-C14 aromatic hydrocarbon group” in the “optionally substitutedC6-C14 aromatic hydrocarbon group” represented by R₂ or R₃ is preferablya phenyl group or a naphthyl group, more preferably a phenyl group.

Examples of the “substituent” for the “optionally substituted C6-C14aromatic hydrocarbon group” represented by R₂ or R₃ include thesubstituent as described above. The substituent is preferably a halogenatom. The optionally halogen atom-substituted C6-C14 aromatichydrocarbon group is preferably an optionally fluorine atom- or chlorineatom-substituted phenyl group, more preferably a phenyl group, afluorophenyl group, a difluorophenyl group, a trifluorophenyl group, achlorophenyl group, a dichlorophenyl group, or a trichlorophenyl group,even more preferably a phenyl group.

The “4- to 8-membered nitrogen-containing saturated heterocyclic group”in the “optionally substituted 4- to 8-membered nitrogen-containingsaturated heterocyclic group” formed by R₂ and R₃, together with thenitrogen atom bonded thereto, is a 4- to 8-membered saturatedheterocyclic group containing at least one nitrogen atom in the ring andfurther containing 0 to 2 identical or different heteroatoms selectedfrom the group consisting of an oxygen atom and a sulfur atom in thering.

The 4- to 8-membered nitrogen-containing saturated heterocyclic group ispreferably a 4- to 8-membered saturated heterocyclic group containingone nitrogen atom, more preferably an azetidinyl group, a pyrrolidinylgroup, or a piperidinyl group.

Examples of the “substituent” for the “optionally substituted 4- to8-membered nitrogen-containing saturated heterocyclic group” include thesubstituent as described above. The substituent is preferably a hydroxylgroup.

The optionally substituted 4- to 8-membered nitrogen-containingsaturated heterocyclic group is preferably an optionally hydroxylgroup-substituted 4- to 8-membered nitrogen-containing saturatedheterocyclic group, more preferably an azetidinyl group, a pyrrolidinylgroup, a piperidinyl group, a hydroxyazetidinyl group, ahydroxypyrrolidinyl group, or a hydroxypiperidinyl group, even morepreferably a pyrrolidinyl group, a piperidinyl group, or ahydroxyazetidinyl group.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I), R₂ and R₃ arepreferably the same as or different from each other and each represent ahydrogen atom, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, or an optionally substitutedC6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionally form,together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup, more preferably are the same as or different from each other andeach represent a hydrogen atom, a C1-C6 alkoxy group, a C1-C6 alkylgroup optionally having a C1-C6 alkoxy group as a substituent, or aC6-C14 aromatic hydrocarbon group optionally having a halogen atom as asubstituent, or R₂ and R₃ optionally form, together with the nitrogenatom bonded thereto, an optionally substituted 4- to 8-memberednitrogen-containing saturated heterocyclic group, even more preferablyare the same as or different from each other and each represent ahydrogen atom, a C1-C6 alkoxy group, a C1-C6 alkyl group, or a C6-C14aromatic hydrocarbon group, or R₂ and R₃ optionally form, together withthe nitrogen atom bonded thereto, a 4- to 8-membered nitrogen-containingsaturated heterocyclic group optionally having a hydroxyl group as asubstituent, further preferably are the same as or different from eachother and each represent a C1-C6 alkoxy group, a C1-C6 alkyl group, or aC6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionally form,together with the nitrogen atom bonded thereto, a 4- to 8-memberednitrogen-containing saturated heterocyclic group optionally having ahydroxyl group as a substituent, still further preferably are the sameas or different from each other and each represent a methoxy group, amethyl group, or a phenyl group, or R₂ and R₃ optionally form, togetherwith the nitrogen atom bonded thereto, an azetidinyl group optionallyhaving a hydroxyl group as a substituent, a pyrrolidinyl group, or apiperidinyl group, still further preferably are the same as or differentfrom each other and each represent a methoxy group, a methyl group, or aphenyl group, or R₂ and R³ optionally form, together with the nitrogenatom bonded thereto, a hydroxyazetidinyl group, a pyrrolidinyl group, ora piperidinyl group, still further preferably each are a methyl group.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I) of the presentinvention, R₄, R₅, and R₆ are the same as or different from each otherand each represent a hydrogen atom or an optionally substituted C1-C6alkyl group.

The “C1-C6 alkyl group” in the “optionally substituted C1-C6 alkylgroup” represented by R₄, R₅, or R₆ is preferably a methyl group, anethyl group, a n-propyl group, an isopropyl group, a n-butyl group, anisobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group,or a hexyl group, more preferably a methyl group, an ethyl group, an-propyl group, an isopropyl group, a n-butyl group, an isobutyl group,a sec-butyl group, or a tert-butyl group, further preferably a methylgroup, an ethyl group, a n-propyl group, or an isopropyl group,particularly preferably a methyl group.

Examples of the “substituent” for the “optionally substituted C1-C6alkyl group” represented by R₄, R₅, or R⁶ include the substituent asdescribed above. The substituent is preferably a dialkylamino group or asaturated heterocyclic group, more preferably a di(C1-C6 alkyl)aminogroup or a 4- to 8-membered saturated heterocyclic group having anitrogen atom, even more preferably a di(C1-C4 alkyl)amino group or a 4-to 8-membered saturated heterocyclic group having a nitrogen atom,further preferably a dimethylamino group, a methylethylamino group, adiethylamino group, a methylisopropylamino group, a 1-piperidinyl group,or a 1-pyrrolidinyl group, particularly preferably a dimethylaminogroup.

The number of the substituent is not particularly limited and ispreferably 1.

The “optionally substituted C1-C6 alkyl group” represented by R₄, R₅, orR₆ is preferably a C1-C6 alkyl group optionally having, as asubstituent, a di(C1-C6 alkyl)amino group or a 4- to 8-memberedsaturated heterocyclic group having a nitrogen atom, more preferably aC1-C4 alkyl group optionally having, as a substituent, a di(C1-C4alkyl)amino group or a 4- to 8-membered saturated heterocyclic grouphaving a nitrogen atom, even more preferably a methyl group, an ethylgroup, a n-propyl group, an isopropyl group, a n-butyl group, anisobutyl group, a sec-butyl group, a tert-butyl group, adimethylaminomethyl group, a methylethylaminomethyl group, adiethylaminomethyl group, a methylisopropylaminomethyl group, adimethylaminoethyl group, a diethylaminoethyl group, a1-piperidinylmethyl group, or a 1-pyrrolidinylmethyl group, furtherpreferably a methyl group, an ethyl group, a n-propyl group, anisopropyl group, a n-butyl group, an isobutyl group, a sec-butyl group,a tert-butyl group, a dimethylaminomethyl group, amethylethylaminomethyl group, a diethylaminomethyl group, amethylisopropylaminomethyl group, a dimethylaminoethyl group, or adiethylaminoethyl group, still further preferably a methyl group, adimethylaminomethyl group, a methylethylaminomethyl group, adiethylaminomethyl group, or a methylisopropylaminomethyl group,particularly preferably a dimethylaminomethyl group.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I), R₄, R₅, and R₆are the same as or different from each other and each are preferably ahydrogen atom or an optionally substituted C1-C6 alkyl group, morepreferably a hydrogen atom or a C1-C6 alkyl group optionally having, asa substituent, a di(C1-C6 alkyl)amino group or a 4- to 8-memberedsaturated heterocyclic group having a nitrogen atom, even morepreferably a hydrogen atom or a C1-C4 alkyl group optionally having, asa substituent, a di(C1-C4 alkyl)amino group or a 4- to 8-memberedsaturated heterocyclic group having a nitrogen atom, further preferablya hydrogen atom, a methyl group, an ethyl group, a n-propyl group, anisopropyl group, a n-butyl group, an isobutyl group, a sec-butyl group,a tert-butyl group, a dimethylaminomethyl group, amethylethylaminomethyl group, a diethylaminomethyl group, amethylisopropylaminomethyl group, a dimethylaminoethyl group, adiethylaminoethyl group, a 1-piperidinylmethyl group, or a1-pyrrolidinylmethyl group, still further preferably a hydrogen atom, amethyl group, an ethyl group, a n-propyl group, an isopropyl group, an-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group,a dimethylaminomethyl group, a methylethylaminomethyl group, adiethylaminomethyl group, a methylisopropylaminomethyl group, adimethylaminoethyl group, or a diethylaminoethyl group, still furtherpreferably a hydrogen atom, a methyl group, a dimethylaminomethyl group,a methylethylaminomethyl group, a diethylaminomethyl group, or amethylisopropylaminomethyl group, particularly preferably a hydrogenatom or a dimethylaminomethyl group.

In the pyrazolo[3,4-d]pyrimidine compound of formula (I),—C(R₄)═C(R₅)(R₆) represented by Y is preferably

particularly preferably

A compound preferred as the pyrazolo[3,4-d]pyrimidine compound offormula (I) is a compound or a salt thereof, wherein X is an optionallysubstituted 4- to 10-membered nitrogen-containing saturated heterocyclicgroup;

Y is —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a halogen atom, a cyano group, a C2-C6 alkenylgroup, an optionally substituted C1-C6 alkoxy group, an optionallysubstituted C1-C5 alkyl group, an optionally substituted amino group, anoptionally substituted C3-C7 cycloalkyl group, a C6-C14 aromatichydrocarbon group, or a 4- to 14-membered unsaturated heterocyclicgroup, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, together with therespective carbon atoms bonded thereto, a benzene ring or a 5- to7-membered saturated or unsaturated heterocyclic ring;W is —CH₂—, an oxygen atom, or —NH—;n is an integer of from 0 to 2;R₁ is an optionally substituted amino group;R₂ and R₃ are the same as or different from each other and each are ahydrogen atom, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, or an optionally substitutedC6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionally form,together with the nitrogen atom bonded thereto, a 4- to 8-memberednitrogen-containing saturated heterocyclic group optionally having ahydroxyl group as a substituent; andR₄, R₅, and R₆ are the same as or different from each other and each area hydrogen atom or an optionally substituted C1-C6 alkyl group.

A compound more preferred as the pyrazolo[3,4-d]pyrimidine compound offormula (I) is a compound or a salt thereof, wherein X is a 4- to10-membered nitrogen-containing saturated heterocyclic group optionallyhaving a halogen atom, a hydroxyl group, a cyano group, a C1-C6 alkylgroup, or an amino group as a substituent;

Y is —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a halogen atom, a cyano group, a C2-C6 alkenylgroup, an optionally substituted C1-C6 alkoxy group, an optionallysubstituted C1-C6 alkyl group, an optionally substituted amino group, anoptionally substituted C3-C7 cycloalkyl group, or a 4- to 14-memberedunsaturated heterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionallyform, together with the respective carbon atoms bonded thereto, abenzene ring or a 5- to 7-membered saturated or unsaturated heterocyclicring;W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group;R₂ and R₃ are the same as or different from each other and each are ahydrogen atom, an optionally substituted C1-C6 alkoxy group, anoptionally substituted C1-C6 alkyl group, or an optionally substitutedC6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionally form,together with the nitrogen atom bonded thereto, a 4- to 8-memberednitrogen-containing saturated heterocyclic group optionally having ahydroxyl group as a substituent; andR₄, R₅, and R₆ are the same as or different from each other and each area hydrogen atom or an optionally substituted C1-C6 alkyl group.

A compound more preferred as the pyrazolo[3,4-d]pyrimidine compound offormula (I) is a compound or a salt thereof, wherein X is a 4- to8-membered nitrogen-containing saturated heterocyclic group optionallyhaving a halogen atom or a C1-C6 alkyl group as a substituent;

Y is —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a halogen atom, a cyano group, a C2-C6 alkenylgroup, an optionally halogen atom-substituted C1-C6 alkoxy group, aC1-C6 alkyl group, an optionally C1-C6 alkyl group-substituted aminogroup, a C3-C7 cycloalkyl group, or a monocyclic 4- to 6-memberedunsaturated heterocyclic group having one oxygen atom, or Z₁ and Z₂, orZ₃ and Z₄ optionally form, together with the respective carbon atomsbonded thereto, a benzene ring or a 5- to 7-membered saturated orunsaturated heterocyclic ring;W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group;R₂ and R₃ are the same as or different from each other and each are ahydrogen atom, a C1-C6 alkoxy group, a C1-C6 alkyl group, or a C6-C14aromatic hydrocarbon group, or R₂ and R₃ optionally form, together withthe nitrogen atom bonded thereto, an optionally hydroxylgroup-substituted 4- to 8-membered nitrogen-containing saturatedheterocyclic group; andR₄, R₅, and R₆ are the same as or different from each other and each area hydrogen atom or an optionally di(C1-C6 alkyl)amino group-substitutedC1-C6 alkyl group.

A compound more preferred as the pyrazolo[3,4-d]pyrimidine compound offormula (I) is a compound or a salt thereof, wherein X is a pyrrolidinylgroup optionally having a C1-C6 alkyl group as a substituent, or apiperidinyl group optionally having a halogen atom as a substituent;

Y is —C(R₄)═C(R₅)(R₆);Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a halogen atom, a cyano group, a C2-C6 alkenylgroup, an optionally halogen atom-substituted C1-C6 alkoxy group, aC1-C6 alkyl group, an optionally C1-C6 alkyl group-substituted aminogroup, a C3-C7 cycloalkyl group, or a monocyclic 4- to 6-memberedunsaturated heterocyclic group having one oxygen atom, or Z₁ and Z₂, orZ₃ and Z₄ optionally form, together with the respective carbon atomsbonded thereto, a benzene ring or a 5- to 7-membered saturated orunsaturated heterocyclic ring;W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group;R₂ and R₃ are the same as or different from each other and each are ahydrogen atom, a C1-C6 alkoxy group, a C1-C6 alkyl group, or a C6-C14aromatic hydrocarbon group, or R₂ and R₃ optionally form, together withthe nitrogen atom bonded thereto, an optionally hydroxylgroup-substituted 4- to 8-membered nitrogen-containing saturatedheterocyclic group; andR₄, R₅, and R₆ are the same as or different from each other and each area hydrogen atom or a dimethylaminomethyl group.

A compound more preferred as the pyrazolo[3,4-d]pyrimidine compound offormula (I) is a compound or a salt thereof, wherein

X is a pyrrolidinyl group, a methylpyrrolidinyl group, a piperidinylgroup, or a fluoropiperidinyl group;

Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group optionally having a fluorineatom as a substituent, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring;W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group; andR₂ and R₃ are the same as or different from each other and each are ahydrogen atom, a methoxy group, a methyl group, or a phenyl group, or R₂and R₃ optionally form, together with the nitrogen atom bonded thereto,an azetidinyl group optionally having a hydroxyl group as a substituent,a pyrrolidinyl group, or a piperidinyl group.

A compound more preferred as the pyrazolo[3,4-d]pyrimidine compound offormula (I) is a compound or a salt thereof, wherein X is a pyrrolidinylgroup, a methylpyrrolidinyl group, a piperidinyl group, or afluoropiperidinyl group;

Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group, a fluoromethoxy group, adifluoromethoxy group, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring;W is —CH₂—, an oxygen atom, or —NH—;n is 0;R₁ is an amino group; andR₂ and R₃ are the same as or different from each other and each are amethoxy group, a methyl group, or a phenyl group, or R₂ and R₃optionally form, together with the nitrogen atom bonded thereto, ahydroxyazetidinyl group, a pyrrolidinyl group, or a piperidinyl group.

A compound more preferred as the pyrazolo[3,4-d]pyrimidine compound offormula (I) is a compound or a salt thereof, wherein X is a pyrrolidinylgroup, a methylpyrrolidinyl group, a piperidinyl group, or afluoropiperidinyl group;

Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group, a fluoromethoxy group, adifluoromethoxy group, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring;W is —CH₂— or an oxygen atom;n is 0;R₁ is an amino group; andR₂ and R₃ are the same as or different from each other and each are amethoxy group, a methyl group, or a phenyl group, or R₂ and R₃optionally form, together with the nitrogen atom bonded thereto, ahydroxyazetidinyl group, a pyrrolidinyl group, or a piperidinyl group.

A compound more preferred as the pyrazolo[3,4-d]pyrimidine compound offormula (I) is a compound or a salt thereof, wherein X is a pyrrolidinylgroup, a methylpyrrolidinyl group, a piperidinyl group, or afluoropiperidinyl group;

Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group, a fluoromethoxy group, adifluoromethoxy group, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring;W is —CH₂— or an oxygen atom;n is 0;R₁ is an amino group; andeach of R₂ and R₃ is a methyl group.

A compound more preferred as the pyrazolo[3,4-d]pyrimidine compound offormula (I) is a compound or a salt thereof, wherein

X is a piperidinyl group or a fluoropiperidinyl group;

Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group, a fluoromethoxy group, adifluoromethoxy group, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring;W is —CH₂— or an oxygen atom;n is 0;R₁ is an amino group; andeach of R₂ and R₃ is a methyl group.

A compound more preferred as the pyrazolo[3,4-d]pyrimidine compound offormula (I) is a compound or a salt thereof, wherein

X is a piperidinyl group;

Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, amethoxy group, a fluoromethoxy group, a difluoromethoxy group, or amethyl group, or Z₁ and Z₂, or Z₃ and Z₄ optionally form, together withthe respective carbon atoms bonded thereto, a benzene ring;

W is —CH₂—;

n is 0;R₁ is an amino group; andeach of R₂ and R₃ is a methyl group.

Specific examples of the pyrazolo[3,4-d]pyrimidine compound of thepresent invention can include, but are not limited to, Synthesis Examplecompounds 1 to 79 produced in Synthesis Examples mentioned later.

Among them, preferred examples of the pyrazolo[3,4-d]pyrimidine compoundcan include the following:

-   (1)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-bromo-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (2)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (3)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (4)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (5)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (6)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (7)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (8)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (9)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (10)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-5-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (11)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (12)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (13)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (14)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (15)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-(difluoromethoxy)-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (16)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-(fluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (17)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-bromo-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (18)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,-   (19)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,    and-   (20)    (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2,5-dichloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.

The pyrazolo[3,4-d]pyrimidine compound is most preferably(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.

Next, the method for producing the pyrazolo[3,4-d]pyrimidine compoundwill be described.

The pyrazolo[3,4-d]pyrimidine compound of formula (I) can be producedby, for example, a production method given below or method shown inSynthesis Examples. However, the method for producing thepyrazolo[3,4-d]pyrimidine compound of formula (I) is not limited tothese reaction examples.

(Production Method 1)

wherein L₁ and L₂ are the same as or different from each other and eachrepresent a leaving group; P₁ represents a protective group for theamino group contained in X; and X, Y, and n are as defined above.

(1st Step)

This step is a step of producing a compound of formula (IV) usingcompounds of formula (II) and the formula (III).

In the formula (II), the leaving group represented by L₁ is preferably abromine atom or an iodine atom. This compound is a commerciallyavailable product or can be produced according to a method known in theart.

In the formula (III), examples of the leaving group of L₂ include achlorine atom, a bromine atom, an iodine atom, methanesulfonate, andp-toluenesulfonate. This compound is a commercially available product orcan be produced according to a method known in the art.

The compound of formula (III) can be used at from 1 to 10 mol,preferably from 1 to 5 mol, with respect to 1 mol of the compound offormula (II).

When the compound of formula (III) is used as an alkylation reagent,this step can be carried out in the presence of a base.

Examples of the base include: inorganic bases such as sodiumbicarbonate, sodium carbonate, potassium carbonate, cesium carbonate,cesium hydroxide, sodium hydride, and potassium hydride; and organicamines such as trimethylamine, triethylamine, tripropylamine,diisopropylethylamine, N-methylmorpholine, pyridine,4-(N,N-dimethylamino)pyridine, lutidine, and collidine. Potassiumcarbonate is preferred.

The amount of the base used can be from 1 to 100 mol, preferably from 2to 10 mol, with respect to 1 mol of the compound of formula (II).

Examples of the solvent include: aprotic polar solvents such asN,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, andN-methylpyrrolidin-2-one; ethers such as tetrahydrofuran and1,4-dioxane; and nitriles such as acetonitrile. These solvents can beused alone or as a mixture. N,N-Dimethylacetamide is preferred.

The reaction time is preferably from 0.1 to 100 hours, more preferablyfrom 0.5 to 24 hours. The reaction temperature is preferably from 0° C.to the boiling temperature of the solvent, more preferably from 0 to100° C.

Thus-obtained compound of formula (IV) can be isolated and purified by aseparation and purification approach known in the art mentioned later orcan be subjected to a subsequent step without being isolated andpurified.

(2nd step)

This step is a step of producing a compound of formula (V) by reactingthe compound of formula (IV) with, for example, a transition metal and,if necessary, a base, in a solvent which has no side reaction in thepresence of an alcohol in a carbon monoxide atmosphere.

In this step, the pressure of the carbon monoxide is usually from 1 atmto 10 atm, preferably from 1 atm to 5 atm.

The amount of the alcohol compound used can be from 1 to 10 mol,preferably from 1 to 5 mol, with respect to 1 mol of the compound offormula (IV). Examples of the alcohol compound include methanol,ethanol, propanol, isopropyl alcohol, diethylaminoethanol, isobutanol,4-(2-hydroxyethyl)morpholine, 3-morpholinopropanol, anddiethylaminopropanol.

The transition metal which may be used in this step is, for example, apalladium catalyst (e.g., palladium acetate,tris(dibenzylideneacetone)dipalladium,bis(triphenylphosphine)palladium(II) dichloride, a1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride-dichloromethane complex). If necessary, a ligand (e.g.,triphenylphosphine, xantphos, tri-tert-butylphosphine) is added.

The amount of the transition metal used differs by the type of thecatalyst and is usually from 0.0001 to 1 mol, preferably from 0.001 to0.5 mol, with respect to 1 mol of the compound of formula (IV). Theamount of the ligand used is usually from 0.0001 to 4 mol, preferablyfrom 0.01 to 2 mol, with respect to 1 mol of the compound of formula(IV).

For the reaction, if necessary, a base can be added. Examples of thebase include: organic bases such as triethylamine,diisopropylethylamine, pyridine, lutidine, collidine,4-dimethylaminopyridine, N-methylmorpholine, potassium tert-butyrate,sodium tert-butyrate, sodium methoxide, sodium ethoxide, lithiumhexamethyldisilazide, sodium hexamethyldisilazide, potassiumhexamethyldisilazide, and butyllithium; and inorganic bases such assodium bicarbonate, sodium carbonate, potassium carbonate, cesiumcarbonate, sodium hydroxide, and sodium hydride.

The amount of the base used is usually from 0.1 to 50 mol, preferablyfrom 1 to 20 mol, with respect to 1 mol of the compound of formula (IV).

The reaction solvent is not particularly limited as long as the solventdoes not interfere with the reaction. Examples thereof includehydrocarbons (e.g., benzene, toluene, xylene), nitriles (e.g.,acetonitrile), ethers (e.g., dimethoxyethane, tetrahydrofuran,1,4-dioxane), alcohols (e.g., methanol, ethanol), aprotic polar solvents(e.g., dimethylformamide, dimethylacetamide, N-methylpyrrolidinone,dimethyl sulfoxide, hexamethylphosphoramide), water, and mixturesthereof.

The reaction time is preferably from 0.1 to 100 hours, more preferablyfrom 0.5 to 24 hours.

The reaction temperature is from 0° C. to the boiling temperature of thesolvent, preferably from 0 to 150° C.

After this reaction, an ester mixture corresponding to the carboxylicacid compound (V) and the alcohol used is formed. Therefore, this estermixture is converted to a compound of formula (V) through hydrolysisreaction.

The hydrolysis reaction is carried out using a base. Examples of thebase include: organic bases such as diethylamine, diisopropylamine,potassium tert-butyrate, sodium tert-butyrate, sodium methoxide, sodiumethoxide, lithium hexamethyldisilazide, sodium hexamethyldisilazide,potassium hexamethyldisilazide, and butyllithium; and inorganic basessuch as sodium bicarbonate, sodium carbonate, potassium carbonate,cesium carbonate, and sodium hydroxide.

The reaction solvent is not particularly limited as long as the solventdoes not interfere with the hydrolysis reaction. Examples thereofinclude hydrocarbons (e.g., benzene, toluene, xylene), nitriles (e.g.,acetonitrile), ethers (e.g., dimethoxyethane, tetrahydrofuran,1,4-dioxane), alcohols (e.g., methanol and ethanol), aprotic polarsolvents (e.g., dimethylformamide, dimethylacetamide,N-methylpyrrolidinone, dimethyl sulfoxide, hexamethylphosphoramide),water, and mixtures thereof.

The hydrolysis reaction time is preferably from 0.1 to 100 hours, morepreferably from 0.5 to 24 hours.

The hydrolysis reaction temperature is from 0° C. to the boilingtemperature of the solvent, preferably from 0 to 150° C.

Thus-obtained compound of formula (V) can be isolated and purified by aseparation and purification approach known in the art mentioned later orcan be subjected to a subsequent step without being isolated andpurified.

(3rd Step)

This step is a step of producing a compound of formula (VI) bydeprotecting the protected amino group in the compound of formula (V).

The deprotection method can be carried out by an ordinary method knownin the art, for example, a method described in Protective Groups inOrganic Synthesis, T. W. Greene, John Wiley & Sons, Inc. (1981) or amethod equivalent thereto. Examples of the protective group includetert-butyloxycarbonyl.

When a tert-butyloxycarbonyl group is used as the protective group, thedeprotection is preferably performed under acidic conditions. Examplesof the acid include hydrochloric acid, acetic acid, trifluoroaceticacid, sulfuric acid, methanesulfonic acid, and tosylic acid.Alternatively, deprotection using Lewis acid is also preferred. Examplesthereof include trimethylsilyl iodide and a boron trifluoride-diethylether complex.

The amount of the acid used is preferably from 1 to 100 mol, withrespect to 1 mol of the compound of formula (V).

The reaction solvent is not particularly limited as long as the solventdoes not interfere with the reaction. Examples of the solvent usedinclude alcohols (e.g., methanol), hydrocarbons (e.g., benzene, toluene,xylene), halogenated hydrocarbons (e.g., methylene chloride, chloroform,1,2-dichloroethane), nitriles (e.g., acetonitrile), ethers (e.g.,dimethoxyethane, tetrahydrofuran), aprotic polar solvents (e.g.,N,N-dimethylformamide, dimethyl sulfoxide, hexamethylphosphoramide), andmixtures thereof.

The reaction time is preferably from 0.1 to 100 hours, more preferablyfrom 0.5 to 24 hours.

The reaction temperature is from 0 to 120° C., preferably from 0 to 90°C.

Thus-obtained compound of formula (VI) can be isolated and purified by aseparation and purification approach known in the art mentioned later orcan be subjected to a subsequent step without being isolated andpurified.

(4th Step)

This step is a step of producing a compound of formula (VII) through thecondensing reaction of the compound of formula (VI) with a carboxylicacid represented by Y—COOH or an acid halide represented by Y—C(═O)-L (Lrepresents a chlorine atom or a bromine atom).

When the carboxylic acid represented by Y—COOH is used as a reagent, thereaction is carried out using from 0.5 to 10 mol, preferably from 1 to 3mol, of the carboxylic acid with respect to 1 mol of the compound offormula (VI) in the presence of an appropriate condensing agent.

This carboxylic acid is a commercially available product or can beproduced according to a method known in the art.

The reaction solvent is not particularly limited as long as the solventdoes not interfere with the reaction. Preferred examples thereofinclude: alcohols such as isopropanol and tert-butyl alcohol;hydrocarbons such as toluene and benzene; halogenated hydrocarbons suchas methylene chloride and chloroform; ethers such as tetrahydrofuran and1,4-dioxane; aprotic polar solvents such as dimethylformamide,dimethylacetamide, N-methylpyrrolidinone, and dimethyl sulfoxide; andmixed solvents thereof.

The reaction temperature is usually from −78 to 200° C., preferably from0 to 50° C.

The reaction time is usually from 5 minutes to 3 days, preferably from 5minutes to 10 hours.

Examples of the condensing agent include diphenylphosphoryl azide,N,N′-dicyclohexylcarbodiimide,benzotriazol-1-yloxy-trisdimethylaminophosphonium salt,4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a combination of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and1-hydroxybenzotriazole, 2-chloro-1,3-dimethylimidazolinium chloride, andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylhexauroniumhexafluorophosphate.

For the reaction, if necessary, a base can be added. Examples of thebase include: organic bases such as triethylamine,diisopropylethylamine, pyridine, lutidine, collidine,4-dimethylaminopyridine, potassium tert-butyrate, sodium tert-butyrate,sodium methoxide, sodium ethoxide, lithium hexamethyldisilazide, sodiumhexamethyldisilazide, potassium hexamethyldisilazide, and butyllithium;and inorganic bases such as sodium bicarbonate, sodium carbonate,potassium carbonate, cesium carbonate, sodium hydroxide, and sodiumhydride.

The amount of the base used is preferably from 1 to 100 mol, morepreferably from 1 to 10 mol, with respect to 1 mol of the compound offormula (VI).

When the acid halide represented by Y—C(═O)-L (L represents a chlorineatom or a bromine atom) is used as a reagent, the reaction is carriedout using from 0.5 to 5 mol, preferably from 0.9 to 1.1 mol, of the acidhalide with respect to 1 mol of the compound of formula (VI).

This acid halide is a commercially available product or can be producedaccording to a method known in the art.

The reaction solvent is not particularly limited as long as the solventdoes not interfere with the reaction. Preferred examples thereofinclude: water; hydrocarbons such as toluene and benzene; halogenatedhydrocarbons such as methylene chloride and chloroform; ethers such astetrahydrofuran and 1,4-dioxane; nitriles such as acetonitrile; aproticpolar solvents such as dimethylformamide, dimethylacetamide, andN-methylpyrrolidinone; and mixed solvents thereof.

The reaction temperature is usually from −78 to 200° C., preferably from−20 to 50° C.

The reaction time is usually from 5 minutes to 3 days, preferably from 5minutes to 10 hours.

For the reaction, if necessary, a base can be added. Examples of thebase include: organic bases such as triethylamine,diisopropylethylamine, pyridine, lutidine, collidine,4-dimethylaminopyridine, potassium tert-butyrate, sodium tert-butyrate,sodium methoxide, sodium ethoxide, lithium hexamethyldisilazide, sodiumhexamethyldisilazide, potassium hexamethyldisilazide, and butyllithium;and inorganic bases such as sodium bicarbonate, sodium carbonate,potassium carbonate, cesium carbonate, sodium hydroxide, and sodiumhydride.

The amount of the base used is preferably from 1 to 100 mol, morepreferably from 1 to 10 mol, with respect to 1 mol of the compound offormula (VI).

Thus-obtained compound of formula (VII) can be isolated and purified bya separation and purification approach known in the art mentioned later.

(Production Method 2)

wherein L₃ represents a leaving group; and X, Y, Z₁, Z₂, Z₃, Z₄, W, n,R₁, R₂, and R₃ are as defined above.

(5th Step)

This step is a step of producing a compound of formula (IX) through thecondensing reaction of a compound of formula (VII) with a compound offormula (VIII).

The reaction is carried out using from 0.5 to 10 mol, preferably from 1to 3 mol, of the compound of formula (VIII) with respect to 1 mol of thecompound of formula (VII) in the presence of an appropriate condensingagent as a reagent. This aniline compound is a commercially availableproduct or can be produced according to a method known in the art.

The reaction solvent is not particularly limited as long as the solventdoes not interfere with the reaction. Preferred examples thereof includeisopropanol, tert-butyl alcohol, toluene, benzene, methylene chloride,chloroform, tetrahydrofuran, 1,4-dioxane, dimethylformamide,dimethylacetamide, N-methylpyrrolidinone, dimethyl sulfoxide, and mixedsolvents thereof.

The reaction temperature is usually from −78 to 200° C., preferably from0 to 50° C.

The reaction time is usually from 5 minutes to 3 days, preferably from 5minutes to 10 hours.

Examples of the condensing agent include diphenylphosphoryl azide,N,N′-dicyclohexylcarbodiimide,benzotriazol-1-yloxy-trisdimethylaminophosphonium salt,4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a combination of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and1-hydroxybenzotriazole, 2-chloro-1,3-dimethylimidazolinium chloride, andO-(7-azabenzotriazol-1-yl)-N,N,N′,N-tetramethylhexauroniumhexafluorophosphate.

For the reaction, if necessary, a base can be added. Examples of thebase include: organic bases such as triethylamine,diisopropylethylamine, pyridine, lutidine, collidine,4-dimethylaminopyridine, potassium tert-butyrate, sodium tert-butyrate,sodium methoxide, sodium ethoxide, lithium hexamethyldisilazide, sodiumhexamethyldisilazide, potassium hexamethyldisilazide, and butyllithium;and inorganic bases such as sodium bicarbonate, sodium carbonate,potassium carbonate, cesium carbonate, sodium hydroxide, and sodiumhydride.

The amount of the base used is from 1 to 100 mol, preferably from 1 to10 mol, with respect to 1 mol of the compound of formula (VII).

Thus-obtained compound of formula (IX) can be isolated and purified by aseparation and purification approach known in the art mentioned later orcan be subjected to a subsequent step without being isolated andpurified.

(6th Step)

This step is a step of producing the pyrazolo[3,4-d]pyrimidine compoundof formula (I) from the compound of formula (IX) and a compound offormula (X).

In the formula (X), the leaving group represented by L₃ is preferablychlorine, bromine, iodine, methanesulfonic acid ester, orp-toluenesulfonic acid ester. The compound of formula (X) is acommercially available product or can be produced according to a methodknown in the art.

The compound of formula (X) can be used at from 1 to 10 mol, preferablyfrom 1 to 5 mol, with respect to 1 mol of the compound of formula (IX).

For the reaction, if necessary, a base can be added. Examples of thebase include: inorganic bases such as sodium bicarbonate, sodiumcarbonate, potassium carbonate, cesium carbonate, cesium hydroxide,sodium hydride, and potassium hydride; and organic amines such astrimethylamine, triethylamine, tripropylamine, diisopropylethylamine,N-methylmorpholine, pyridine, 4-(N,N-dimethylamino)pyridine, lutidine,and collidine.

The amount of the base used can be from 1 to 100 mol, preferably from 2to 10 mol, with respect to 1 mol of the compound of formula (IX).

Examples of the solvent which can be used include N,N-dimethylformamide,N,N-dimethylacetamide, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane,N-methylpyrrolidin-2-one, acetonitrile, and mixed solvents thereof.

The reaction time is preferably from 0.1 to 100 hours, more preferablyfrom 0.5 to 24 hours.

The reaction temperature is from 0° C. to the boiling temperature of thesolvent, preferably from 0 to 100° C.

Thus-obtained pyrazolo[3,4-d]pyrimidine compound of formula (I) can beisolated and purified by a separation and purification approach known inthe art mentioned later.

Also, the pyrazolo[3,4-d]pyrimidine compound (I) can be produced by, forexample, the following production method:

(Production Method 3)

wherein Z₁, Z₂, Z³, Z₄, W, R₂, and R₃ are as defined above.

(7th Step)

This step is a step of producing a compound of formula (XIII) throughthe condensing reaction of a compound of formula (XI) with a compound offormula (XII).

The reaction is carried out using from 0.5 to 10 mol, preferably from 1to 3 mol, of the compound of formula (XII) with respect to 1 mol of thecompound of formula (XI) in the presence of an appropriate condensingagent as a reagent. This amine (XII) is a commercially available productor can be produced according to a method known in the art.

The reaction solvent is not particularly limited as long as the solventdoes not interfere with the reaction. Preferred examples thereof includeisopropanol, tert-butyl alcohol, toluene, benzene, methylene chloride,chloroform, tetrahydrofuran, 1,4-dioxane, dimethylformamide,dimethylacetamide, N-methylpyrrolidinone, dimethyl sulfoxide, and mixedsolvents thereof.

The reaction temperature is usually from −78 to 200° C., preferably from0 to 50° C.

The reaction time is usually from 5 minutes to 3 days, preferably from 5minutes to 10 hours.

Examples of the condensing agent include diphenylphosphoryl azide,N,N′-dicyclohexylcarbodiimide,benzotriazol-1-yloxy-trisdimethylaminophosphonium salt,4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a combination of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and1-hydroxybenzotriazole, 2-chloro-1,3-dimethylimidazolinium chloride, andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylhexauroniumhexafluorophosphate.

For the reaction, if necessary, a base can be added. Examples of thebase include: organic bases such as triethylamine,diisopropylethylamine, pyridine, lutidine, collidine,4-dimethylaminopyridine, potassium tert-butyrate, sodium tert-butyrate,sodium methoxide, sodium ethoxide, lithium hexamethyldisilazide, sodiumhexamethyldisilazide, potassium hexamethyldisilazide, and butyllithium;and inorganic bases such as sodium bicarbonate, sodium carbonate,potassium carbonate, cesium carbonate, sodium hydroxide, and sodiumhydride.

The amount of the base used is from 1 to 100 mol, preferably from 1 to10 mol, with respect to 1 mol of the compound of formula (XI).

Thus-obtained compound of formula (XIII) can be isolated and purified bya separation and purification approach known in the art mentioned lateror can be subjected to a subsequent step without being isolated andpurified.

(8th Step)

This step is a step of producing a compound of formula (XIV) through thereduction reaction of the compound of formula (XIII) using a metalcatalyst.

The metal catalyst for use in this step is an iron reagent, a palladiumreagent, a nickel reagent, or the like. Hydrogen gas, formic acid, orthe like can be used as a reducing agent.

The reaction solvent is not particularly limited as long as the solventdoes not interfere with the reaction. Examples thereof include alcohols(e.g., methanol, ethanol), ethers (e.g., tetrahydrofuran, 1,4-dioxane),aprotic polar solvents (e.g., dimethylformamide, dimethylacetamide,N-methylpyrrolidinone, dimethyl sulfoxide, hexamethylphosphoramide),water, and mixtures thereof.

The reaction time is preferably from 0.1 to 100 hours, more preferablyfrom 0.5 to 24 hours.

The reaction temperature is from 0° C. to the boiling temperature of thesolvent, preferably from 0 to 150° C.

Thus-obtained compound of formula (XIV) can be isolated and purified bya separation and purification approach known in the art mentioned later.

(Production Method 4)

wherein L₃ represents a leaving group; and Z₁, Z₂, Z₃, Z₄, W, R₂, and R₃are as defined above.

(9th Step)

This step is a step of producing a compound of formula (XIII) from acompound of formula (XV) and a compound of formula (X).

The compound of formula (X) can be used at from 1 to 10 mol, preferablyfrom 1 to 5 mol, with respect to 1 mol of the compound of formula (XV).

Examples of the reagent for use in the reaction include dialkylcarbamoylchloride, S-methyl dialkylcarbamoyl ester, and dichloromethylenedialkyliminium chloride. These reagents are commercially availableproducts or can be produced according to a method known in the art.

The reagent can be used at from 1 to 100 mol, preferably from 2 to 10mol, with respect to 1 mol of the compound of formula (XV).

For the reaction, if necessary, a base can be added. Examples of thebase include: inorganic bases such as sodium bicarbonate, sodiumcarbonate, potassium carbonate, cesium carbonate, cesium hydroxide,sodium hydride, and potassium hydride; and organic amines such astrimethylamine, triethylamine, tripropylamine, diisopropylethylamine,N-methylmorpholine, pyridine, 4-(N,N-dimethylamino)pyridine, lutidine,and collidine.

The amount of the base used can be from 1 to 100 mol, preferably from 2to 10 mol, with respect to 1 mol of the compound of formula (XV).

Examples of the reaction solvent which can be used includeN,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide,tetrahydrofuran, 1,4-dioxane, N-methylpyrrolidin-2-one, acetonitrile,and mixed solvents thereof.

The reaction time is preferably from 0.1 to 100 hours, more preferablyfrom 0.5 to 24 hours.

The reaction temperature is from 0° C. to the boiling temperature of thesolvent, preferably from 0 to 100° C.

Thus-obtained compound of formula (XIII) can be isolated and purified bya separation and purification approach known in the art mentioned lateror can be subjected to a subsequent step without being isolated andpurified.

(10th Step)

This step is a step of producing a compound of formula (XIV) throughreduction reaction from the compound of formula (XIII). This compoundcan be produced by the same production method as in the 8th stepdescribed above.

(Production Method 5)

wherein X, Z₁, Z₂, Z₃, Z₄, W, n, R₁, R₂, and R³ are as defined above.

(11th Step)

This step is a step of producing the pyrazolo[3,4-d]pyrimidine compoundof formula (I) through the condensing reaction of a compound of formula(VII) with a compound of formula (XIV).

The reaction is carried out using from 0.5 to 10 mol, preferably from 1to 3 mol, of the compound of formula (XIV) with respect to 1 mol of thecompound of formula (VII) in the presence of an appropriate condensingagent as a reagent.

The reaction solvent is not particularly limited as long as the solventdoes not interfere with the reaction. Preferred examples thereof includeisopropanol, tert-butyl alcohol, toluene, benzene, methylene chloride,chloroform, tetrahydrofuran, 1,4-dioxane, dimethylformamide,dimethylacetamide, N-methylpyrrolidinone, dimethyl sulfoxide, and mixedsolvents thereof.

The reaction temperature is usually from −78 to 200° C., preferably from0 to 50° C.

The reaction time is usually from 5 minutes to 3 days, preferably from 5minutes to 10 hours.

Examples of the condensing agent include diphenylphosphoryl azide,N,N′-dicyclohexylcarbodiimide,benzotriazol-1-yloxy-trisdimethylaminophosphonium salt,4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a combination of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and1-hydroxybenzotriazole, 2-chloro-1,3-dimethylimidazolinium chloride, andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylhexauroniumhexafluorophosphate.

For the reaction, if necessary, a base can be added. Examples of thebase include: organic bases such as triethylamine,diisopropylethylamine, pyridine, lutidine, collidine,4-dimethylaminopyridine, potassium tert-butyrate, sodium tert-butyrate,sodium methoxide, sodium ethoxide, lithium hexamethyldisilazide, sodiumhexamethyldisilazide, potassium hexamethyldisilazide, and butyllithium;and inorganic bases such as sodium bicarbonate, sodium carbonate,potassium carbonate, cesium carbonate, sodium hydroxide, and sodiumhydride.

The amount of the base used is from 1 to 100 mol, preferably from 1 to10 mol, with respect to 1 mol of the compound of formula (VII).

Thus-obtained pyrazolo[3,4-d]pyrimidine compound of formula (I) can beisolated and purified by a separation and purification approach known inthe art mentioned later.

In Production methods 1 to 5 described above, when it comes to an aminogroup, an imino group, a hydroxy group, a carboxyl group, a carbonylgroup and an amide group, and a functional group having an active protonsuch as indole, a reagent protected in an appropriate step in eachproduction method may be used, or a protective group thereof may beintroduced to the functional group by a conventional method and then theprotective group may be removed.

The “protective group for the amino group or the imino group” is notparticularly limited as long as the protective group has the function.Examples thereof include: aralkyl groups such as a benzyl group, ap-methoxybenzyl group, a 3,4-dimethoxybenzyl group, a o-nitrobenzylgroup, a p-nitrobenzyl group, a benzhydryl group, a trityl group, and acumyl group; lower alkanoyl groups such as a formyl group, an acetylgroup, a propionyl group, a butyryl group, a pivaloyl group, atrifluoroacetyl group, and a trichloroacetyl group; a benzoyl group;arylalkanoyl groups such as a phenylacetyl group and a phenoxyacetylgroup; lower alkoxycarbonyl groups such as a methoxycarbonyl group, anethoxycarbonyl group, a propyloxycarbonyl group, and atert-butoxycarbonyl group; aralkyloxycarbonyl groups such as ap-nitrobenzyloxycarbonyl group and a phenethyloxycarbonyl group; loweralkylsilyl groups such as a trimethylsilyl group and atert-butyldimethylsilyl group; a tetrahydropyranyl group; atrimethylsilylethoxymethyl group; lower alkylsulfonyl groups such as amethylsulfonyl group, an ethylsulfonyl group, and a tert-butylsulfonylgroup; lower alkylsulfinyl groups such as a tert-butylsulfinyl group;arylsulfonyl groups such as a benzenesulfonyl group and atoluenesulfonyl group; and imide groups such as a phthalimide group.Particularly, a trifluoroacetyl group, an acetyl group, atert-butoxycarbonyl group, a benzyloxycarbonyl group, atrimethylsilylethoxymethyl group, or a cumyl group is preferred.

The “protective group for the hydroxy group” is not particularly limitedas long as the protective group has the function. Examples thereofinclude: lower alkyl groups such as a methyl group, an ethyl group, apropyl group, an isopropyl group, and a tert-butyl group; loweralkylsilyl groups such as a trimethylsilyl group and atert-butyldimethylsilyl group; lower alkoxymethyl groups such as amethoxymethyl group and a 2-methoxyethoxymethyl group; atetrahydropyranyl group; a trimethylsilylethoxymethyl group; aralkylgroups such as a benzyl group, a p-methoxybenzyl group, a2,3-dimethoxybenzyl group, a o-nitrobenzyl group, a p-nitrobenzyl group,and a trityl group; and acyl groups such as a formyl group, an acetylgroup, and a trifluoroacetyl group. Particularly, a methyl group, amethoxymethyl group, a tetrahydropyranyl group, atrimethylsilylethoxymethyl group, a tert-butyldimethylsilyl group, or anacetyl group is preferred.

The “protective group for the carboxyl group” is not particularlylimited as long as the protective group has the function. Examplesthereof include: lower alkyl groups such as a methyl group, an ethylgroup, a propyl group, an isopropyl group, and a tert-butyl group;halo-lower alkyl groups such as a 2,2,2-trichloroethyl group; loweralkenyl groups such as an allyl group; a trimethylsilylethoxymethylgroup; and aralkyl groups such as a benzyl group, a p-methoxybenzylgroup, a p-nitrobenzyl group, a benzhydryl group, and a trityl group.Particularly, a methyl group, an ethyl group, a tert-butyl group, anallyl group, a benzyl group, a p-methoxybenzyl group, or atrimethylsilylethoxymethyl group is preferred.

The “protective group for the carbonyl group” is not particularlylimited as long as the protective group has the function. Examplesthereof include ketals and acetals such as ethylene ketal, trimethyleneketal, dimethyl ketal, ethylene acetal, trimethylene acetal, anddimethyl acetal.

The “protective group for the amide group or the functional group havingactive proton such as indole” is not particularly limited as long as theprotective group has the function. Examples thereof include: lower alkylgroups such as a methyl group, an ethyl group, a propyl group, anisopropyl group, and a tert-butyl group; lower alkylsilyl groups such asa trimethylsilyl group and a tert-butyldimethylsilyl group; loweralkoxymethyl groups such as a methoxymethyl group and a2-methoxyethoxymethyl group; a tetrahydropyranyl group; atrimethylsilylethoxymethyl group; aralkyl groups such as a benzyl group,a p-methoxybenzyl group, a 2,3-dimethoxybenzyl group, a o-nitrobenzylgroup, a p-nitrobenzyl group, and a trityl group; and acyl groups suchas a formyl group, an acetyl group, and a trifluoroacetyl group.Particularly, a methyl group, a methoxymethyl group, a tetrahydropyranylgroup, a trimethylsilylethoxymethyl group, a tert-butyldimethylsilylgroup, or an acetyl group is preferred.

The method for removing the protective group differs by the type of theprotective group, the stability of the compound of interest, etc., andis carried out according to, for example, a method described in theliterature (see Protective Groups in Organic Synthesis, 3rd edition, T.W. Greene, John Wiley & Sons, Inc., 1999) or a method equivalentthereto, for example, solvolysis using an acid or a base, i.e., a methodof allowing, for example, 0.01 mol to a large excess of an acid,preferably trifluoroacetic acid, formic acid, or hydrochloric acid, oran equimolar amount to a large excess of a base, preferably potassiumhydroxide or calcium hydroxide, to act thereon; chemical reduction usinga metal halide complex or the like; or catalytic reduction using apalladium-carbon catalyst, a Raney nickel catalyst, or the like.

The pyrazolo[3,4-d]pyrimidine compound of formula (I) and theintermediates thereof can each be isolated and purified easily by aseparation and purification approach known in the art. Examples of suchan approach can include concentration, concentration under reducedpressure, solvent extraction, recrystallization, reprecipitation,preparative reverse-phase high-performance liquid chromatography, columnchromatography, and preparative thin-layer chromatography.

When the pyrazolo[3,4-d]pyrimidine compound of formula (I) has opticalisomers, stereoisomers, tautomers, or rotational isomers, any of theseisomers and mixtures thereof are also included in the compound. Aracemate of the pyrazolo[3,4-d]pyrimidine compound or optically activeforms resolved from the racemate are also included in the compound.These isomers can each be obtained as a single compound by a synthesisapproach and a separation approach (concentration, solvent extraction,column chromatography, recrystallization, etc.) known per se in the art.

The pyrazolo[3,4-d]pyrimidine compound of formula (I) or a salt thereofmay be crystalline. A single crystal form or a polymorphic mixture isalso included in the compound or the salt thereof. The crystals can beproduced by crystallization using a crystallization method known per sein the art. The pyrazolo[3,4-d]pyrimidine compound or the salt thereofmay be a solvate (e.g., a hydrate) or may be a non-solvate. Both of themare also included in the compound or the salt thereof. A compoundlabeled with an isotope (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I) or the like is alsoincluded in the pyrazolo[3,4-d]pyrimidine compound or the salt thereof.

A prodrug of the pyrazolo[3,4-d]pyrimidine compound of formula (I) orthe salt thereof is also encompassed by the compound or the saltthereof. The prodrug refers to a compound which is converted to thepyrazolo[3,4-d]pyrimidine compound or the salt thereof through reactionwith an enzyme, gastric juice, or the like under physiologicalconditions in vivo, i.e., a compound which is converted to thepyrazolo[3,4-d]pyrimidine compound or the salt thereof through enzymaticoxidation, reduction, hydrolysis, etc., or a compound which is convertedto the pyrazolo[3,4-d]pyrimidine compound or the salt thereof throughhydrolysis, etc., with gastric juice or the like. Alternatively, theprodrug of the pyrazolo[3,4-d]pyrimidine compound or the salt thereofmay be converted to the pyrazolo[3,4-d]pyrimidine compound or the saltthereof under physiological conditions as described in Hirokawa-ShotenLtd. (1990) “Iyakuhin no Kaihatsu (Development of drugs in English)”,Vol. 7, Molecular Design, p. 163-198.

The salt of the pyrazolo[3,4-d]pyrimidine compound of formula (I) is notparticularly limited as long as the salt is pharmaceutically acceptable.The salt of the compound of the present invention means a salt routinelyused in the organic chemical field. Examples thereof can include saltsincluding: when the compound has a carboxyl group, a base-addition saltof the carboxyl group; and when the compound has an amino group or abasic heterocyclic group, an acid-addition salt of the amino group orthe basic heterocyclic group.

Examples of the base-addition salt include: alkali metal salts such assodium salt and potassium salt; alkaline earth metal salts such ascalcium salt and magnesium salt; ammonium salts; and organic amine saltssuch as trimethylamine salt, triethylamine salt, dicyclohexylamine salt,ethanolamine salt, diethanolamine salt, triethanolamine salt, procainesalt, and N,N′-dibenzylethylenediamine salt.

Examples of the acid-addition salt include: inorganic acid salts such ashydrochloride, sulfate, nitrate, phosphate, and perchlorate; organicacid salts such as acetate, formate, maleate, fumarate, tartrate,citrate, ascorbate, and trifluoroacetate; and sulfonates such asmethanesulfonate, isethionate, benzenesulfonate, and p-toluenesulfonate.

As shown in Examples mentioned later, combined use of thepyrazolo[3,4-d]pyrimidine compound of formula (I) or the salt thereofwith other antitumor agent(s) enhances the antitumor effect. On theother hand, adverse effects such as toxicity are prevented.

Thus, in one embodiment, the present invention provides an antitumoragent comprising a pyrazolo[3,4-d]pyrimidine compound of formula (I) ora salt thereof and other antitumor agent(s) in combination. In anotherembodiment, the present invention provides an agent enhancing anantitumor effect of other antitumor agent(s), the agent comprising apyrazolo[3,4-d]pyrimidine compound of formula (I) or a salt thereof asan active ingredient. The present invention also provides an agentenhancing an antitumor effect of a pyrazolo[3,4-d]pyrimidine compound offormula (I) or a salt thereof, the agent comprising other antitumoragent(s) as an active ingredient.

Examples of the other antitumor agent(s) include, but are notparticularly limited to, antimetabolites, molecular targeting drugs,platinum-based drugs, and plant alkaloid-based drugs.

Examples of the antimetabolite include 5-fluorouracil (5-FU),5-fluoro-2′-deoxyuridine (FdUrd), tegafur, tegafur-uracil (e.g., UFT),tegafur-gimeracil-oteracil (e.g., TS-1), trifluridine,trifluridine-tipiracil hydrochloride (e.g., Lonsurf), gemcitabine,capecitabine, and cytarabine. The antimetabolite is preferably5-fluorouracil (5-FU), 5-fluoro-2′-deoxyuridine (FdUrd), trifluridine,gemcitabine, or capecitabine.

Examples of the molecular targeting drug include ErbB family (EGFR,Her2, Her3, and Her4) inhibitors, PI3K/AKT/mTOR inhibitors, and CDK4/6inhibitors.

The ErbB family (EGFR, Her2, Her3, and Her4) inhibitor is preferably aHer2 inhibitor, more preferably an anti-Her2 antibody such astrastuzumab, pertuzumab, or trastuzumab emtansine, even more preferablytrastuzumab or trastuzumab emtansine, etc.

The PI3K/AKT/mTOR inhibitor is a compound inhibiting the signalingpathway of PI3K/AKT/mTOR. Examples thereof include AZD5363, AZD8055,everolimus, dactolisib, buparlisib, taselisib, MK2206, andtrans-3-amino-1-methyl-3-[4-(3-phenyl-5H-imidazo[1,2-c]pyrido[3,4-e][1,3]oxazin-2-yl)phenyl]-cyclobutanol.The PI3K/AKT/mTOR inhibitor is preferably AZD8055, everolimus,dactolisib, buparlisib, or taselisib.

Examples of the CDK4/6 inhibitor include palbociclib and abemaciclib.The CDK4/6 inhibitor is preferably palbociclib.

Examples of the platinum-based drug include oxaliplatin, carboplatin,cisplatin, and nedaplatin. The platinum-based drug is preferablycisplatin.

Examples of the plant alkaloid-based drug include microtubule inhibitorssuch as paclitaxel, docetaxel, vinblastine, vincristine, vindesine,vinorelbine, and eribulin, and topoisomerase inhibitors such asirinotecan, nogitecan, and etoposide. The plant alkaloid-based drug ispreferably a microtubule inhibitor such as paclitaxel, docetaxel,vinblastine, vincristine, vindesine, vinorelbine, or eribulin, morepreferably a taxane-based microtubule inhibitor such as paclitaxel ordocetaxel, even more preferably paclitaxel or docetaxel, furtherpreferably paclitaxel.

The other antitumor agent(s) is preferably an antimetabolite, amolecular targeting drug, a platinum-based drug, or a plantalkaloid-based drug, more preferably an antimetabolite, an ErbB family(EGFR, Her2, Her3, and Her4) inhibitor, a PI3K/AKT/mTOR inhibitor, aCDK4/6 inhibitor, a platinum-based drug, or a plant alkaloid-based drug,even more preferably an antimetabolite, a Her2 inhibitor, aPI3K/AKT/mTOR inhibitor, a CDK4/6 inhibitor, a platinum-based drug, or aplant alkaloid-based drug, further preferably an antimetabolite, a Her2inhibitor, a PI3K/AKT/mTOR inhibitor, a CDK4/6 inhibitor, aplatinum-based drug, or a microtubule inhibitor, still furtherpreferably an antimetabolite, a Her2 inhibitor, a PI3K/AKT/mTORinhibitor, a CDK4/6 inhibitor, a platinum-based drug, or a taxane-basedmicrotubule inhibitor, even further preferably 5-fluorouracil (5-FU),5-fluoro-2′-deoxyuridine (FdUrd), tegafur, tegafur-uracil (e.g., UFT),tegafur-gimeracil-oteracil (e.g., TS-1), trifluridine,tritluridine-tipiracil hydrochloride (e.g., Lonsurf), gemcitabine,capecitabine, cytarabine, trastuzumab, pertuzumab, trastuzumabemtansine, AZD5363, AZD8055, everolimus, dactolisib, buparlisib,taselisib, MK2206,trans-3-amino-1-methyl-3-[4-(3-phenyl-5H-imidazo[1,2-c]pyrido[3,4-e][1,3]oxazin-2-yl)phenyl]-cyclobutanol,palbociclib, abemaciclib, oxaliplatin, carboplatin, cisplatin,nedaplatin, paclitaxel, or docetaxel, yet further preferably5-fluorouracil (5-FU), 5-fluoro-2′-deoxyuridine (FdUrd), trifluridine,gemcitabine, capecitabine, trastuzumab, trastuzumab emtansine, AZD8055,everolimus, dactolisib, buparlisib, taselisib, palbociclib, cisplatin,or paclitaxel, particularly preferably capecitabine, trastuzumab, ortrastuzumab emtansine.

The other antitumor agent(s) also includes DDS preparations of theseantitumor agents. Examples of the DDS preparation of “paclitaxel”include albumin-bound paclitaxel (e.g., Abraxane) and paclitaxel micelle(e.g., NK105). Examples of the DDS preparation of “cisplatin” includecisplatin micelle (e.g., NC-6004).

In this context, in the present specification, the term “HER2” includeshuman or nonhuman mammalian HER2 and is preferably human HER2. The term“HER2” also includes isoforms.

The cancer targeted according to the present invention is notparticularly limited within a range which produces an effect ofenhancing an antitumor effect, and is preferably cancer on which thepyrazolo[3,4-d]pyrimidine compound of formula (I) or the salt thereofexerts an antitumor effect, more preferably malignant tumor involvingHER2. In this context, examples of the “malignant tumor involving HER2”include malignant tumor in which the functional deletion, suppression,and/or inhibition of HER2 leads to reduction in incidence, remission ofsymptoms, alleviation, and/or complete cure.

Examples of such a malignant tumor is preferably malignant tumor havingHER2 overexpression, HER2 gene amplification, or HER2 mutation. Examplesof the targeted “malignant tumor” include, but are not particularlylimited to, head and neck cancer, esophageal cancer, stomach cancer,colon cancer, rectal cancer, liver cancer, gallbladder or bile ductcancer, biliary cancer, pancreatic cancer, lung cancer, breast cancer,ovary cancer, uterine cervical cancer, uterine body cancer, kidneycancer, bladder cancer, prostate cancer, testis tumor, bone or softtissue sarcoma, blood cancer, multiple myeloma, skin cancer, braintumor, and mesothelioma. The malignant tumor is preferably breastcancer, stomach cancer, esophageal cancer, ovary cancer, lung cancer,gallbladder or bile duct cancer, biliary cancer, bladder cancer, orcolon cancer, more preferably breast cancer, stomach cancer, esophagealcancer, biliary cancer, ovary cancer, or lung cancer, even morepreferably breast cancer or stomach cancer.

For use of the pyrazolo[3,4-d]pyrimidine compound of formula (I) or thesalt thereof and other antitumor agent(s) as a medicament, variousdosage forms, if necessary, containing pharmaceutical carriers can beadopted according to the prophylactic or therapeutic purposes. The formmay be, for example, any of oral agents, injections, suppositories,ointments, and patches. These dosage forms can each be produced by aroutine formulation method known to those skilled in the art.

The schedule of administration of the pyrazolo[3,4-d]pyrimidine compoundof formula (I) or the salt thereof and the other antitumor agent(s) isappropriately selected within a range in which each active ingredientexerts an antitumor effect. These active ingredients are administeredconcurrently or separately in a staggered manner. In the case ofseparately administering these active ingredients, either of the activeingredients may be administered first.

The pyrazolo[3,4-d]pyrimidine compound of formula (I) or the saltthereof and the other antitumor agent(s) may be prepared in a pluralityof formulations respectively comprising the active ingredients or may beprepared together in one formulation, on the basis of the dosage form ofeach active ingredient, or the schedule of administration. Also, therespective preparations may be produced and distributed together in onepackage suitable for combined use, or may be produced and distributed inseparate packages.

Various organic or inorganic carrier substances routinely used aspharmaceutical materials are used as the pharmaceutical carriers. Asolid preparation is supplemented with an excipient, a binder, adisintegrant, a lubricant, a coating agent, or the like, and a liquidpreparation is supplemented with a solvent, a solubilizer, a suspendingagent, a tonicity agent, a pH adjuster or a buffer, a soothing agent, orthe like. If necessary, pharmaceutical additives such as an antiseptic,an antioxidant, a colorant, a corrigent, and a stabilizer can also beused.

Examples of the excipient include lactose, saccharose, D-mannitol,starch, crystalline cellulose, and calcium silicate.

Examples of the binder include hydroxypropylcellulose, methylcellulose,polyvinylpyrrolidone, maltose syrup powder, and hypromellose.

Examples of the disintegrant include sodium starch glycolate, carmellosecalcium, croscarmellose sodium, crospovidone, low-substitutedhydroxypropylcellulose, and partially pregelatinized starch.

Examples of the lubricant include talc, magnesium stearate, sucrosefatty acid ester, stearic acid, and sodium stearyl fumarate.

Examples of the coating agent include ethylcellulose, aminoalkylmethacrylate copolymer RS, hypromellose, and saccharose.

Examples of the solvent include water, propylene glycol, and saline.

Examples of the solubilizer include polyethylene glycol, ethanol,α-cyclodextrin, macrogol 400, and polysorbate 80.

Examples of the suspending agent include carrageenan, crystallinecellulose-carmellose sodium, and polyoxyethylene hydrogenated castoroil.

Examples of the tonicity agent include sodium chloride, glycerin, andpotassium chloride.

Examples of the pH adjuster or the buffer include sodium citrate,hydrochloric acid, lactic acid, phosphoric acid, and sodium dihydrogenphosphate.

Examples of the soothing agent include procaine hydrochloride andlidocaine.

Examples of the antiseptic include ethyl p-hydroxybenzoate, cresol, andbenzalkonium chloride.

Examples of the antioxidant include sodium sulfite, ascorbic acid, andnatural vitamin E.

Examples of the colorant include titanium oxide, iron sesquioxide, FoodBlue No. 1, and copper chlorophyll.

Examples of the corrigent include aspartame, saccharin, sucralose,1-menthol, and mint flavor.

Examples of the stabilizer include sodium pyrosulfite, sodium edetate,erythorbic acid, magnesium oxide, and dibutylhydroxytoluene.

An oral solid preparation can be prepared by using an excipient, abinder, a disintegrant, a lubricant, a colorant, a corrigent, or thelike and producing tablets, coated tablets, granules, powders, capsules,or the like by a routine method.

An injection can be prepared by using a pH adjuster, a buffer, astabilizer, a tonicity agent, a local anesthetic, or the like andproducing subcutaneous, intramuscular, and intravenous injections by aroutine method.

The amount of the pyrazolo[3,4-d]pyrimidine compound or the salt thereofto be contained in each dosage unit form described above varies by thesymptoms of a patient to receive this or by the dosage form, etc., andis generally desirably from 0.05 to 1000 mg for oral agents, from 0.01to 500 mg for injections, or from 1 to 1000 mg for suppositories, perdosage unit form.

The ratio between the pyrazolo[3,4-d]pyrimidine compound or the saltthereof and one other antitumor agent contained is not particularlylimited within a range which exerts an effect of enhancing an antitumoreffect. The pyrazolo[3,4-d]pyrimidine compound or the salt thereof canbe used at 0.001 to 100 mol per 1 mol of one other antitumor agent.

The daily dose of the pyrazolo[3,4-d]pyrimidine compound or the saltthereof having the dosage form differs by the symptoms, body weight,age, sex, etc., of the patient and cannot be generalized. The daily dosecan be usually from 0.05 to 5000 mg, preferably from 0.1 to 1000 mg, ofthe pyrazolo[3,4-d]pyrimidine compound in an adult (body weight: 50 kg),and this dose is preferably administered once a day or in approximatelytwo or three divided portions per day.

The daily dose of the other antitumor agent(s) having the dosage formdiffers by the type of the antitumor agent used, the formulationthereof, the symptoms, body weight, age, sex, etc., of the patient andcannot be generalized. The daily dose can be usually, for example, from0.05 to 5000 mg, preferably from 0.1 to 1000 mg, of the compound in anadult (body weight: 50 kg), and this dose can preferably be administeredonce a day or in approximately two or three divided portions per day.

Alternatively, the daily dose of the other antitumor agent(s) having thedosage form can be, for example, a dose from 10 to 100%, preferably from50 to 100%, more preferably of 100%, of the recommended dose of theother antitumor agent(s) to be administered alone.

In the present invention, the “recommended dose” is determined by aclinical trial or the like and is a dose which brings about the maximumtherapeutic effect within a range safe to be able to use withoutdeveloping severe adverse effects. Specific examples thereof includedoses approved, recommended, or advised by public institutions such asthe Pharmaceuticals and Medical Devices Agency (PMDA), the Food and DrugAdministration (FDA), and the European Medicines Agency (EMA) anddescribed in package inserts, interview forms, guidelines for treatment,or the like. A dose approved by any of the public institutions PMDA,FDA, and EMA is preferred.

The present invention also relates to a kit preparation comprising thepyrazolo[3,4-d]pyrimidine compound of the present invention or the saltthereof, and a usage instruction stating that thepyrazolo[3,4-d]pyrimidine compound of the present invention or the saltthereof is used in combined administration with other antitumor agent(s)to a cancer patient. In this context, the “usage instruction” can be anyusage instruction stating the dose and preferably recommends the dose,irrespective of the presence or absence of legal binding power. Specificexamples thereof include package inserts and pamphlets. The kitpreparation comprising the usage instruction may be a kit preparationwith the usage instruction printed on or attached to a package, or maybe a kit preparation with the usage instruction included together withthe antitumor agent in a package.

EXAMPLES

Hereinafter, the present invention will be described furtherspecifically with reference to Synthesis Examples and Examples. However,the present invention is not intended to be limited to these Examples byany means.

Synthesis Example

Various types of reagents were used in Synthesis Examples which werecommercially available unless otherwise specified. Purif-Pack® SImanufactured by Moritex Corp., KP-Sil® Silica Prepacked Columnmanufactured by Biotage Japan Ltd., or HP-Sil® Silica Prepacked Columnmanufactured by Biotage Japan Ltd. was used in silica gel columnchromatography.

Purif-Pack® NH manufactured by Moritex Corp. or KP-NH® Prepacked Columnmanufactured by Biotage Japan Ltd. was used in basic silica gel columnchromatography.

Kieselgel™ 60F254, Art. 5744 manufactured by Merck KGaA or NH2 SilicaGel 60F254 Plate manufactured by Wako Pure Chemical Industries, Ltd. wasused in preparative thin-layer chromatography.

NMR spectra were measured using AL400 (400 MHz; JEOL Ltd.), Mercury 400(400 MHz; Agilent Technologies, Inc.) spectrometer, or Inova 400 (400MHz; Agilent Technologies, Inc.) spectrometer equipped with OMNMR probe(Protasis Corp.) and using tetramethylsilane as an internal standard inthe case of containing tetramethylsilane in a deuterated solvent and aNMR solvent as an internal standard in other cases. Total 5 values wereindicated by ppm.

Microwave reaction was carried out using Discover S class manufacturedby CEM Corp.

LCMS spectra were measured using ACQUITY SQD (quadrupole type)manufactured by Waters Corp. under the following conditions:

Column: YMC-Triart C18 manufactured by YMC Co., Ltd., 2.0×50 mm,1.9 μmMS detection: ESI positiveUV detection: 254 and 210 nmColumn flow rate: 0.5 mL/minMobile phase: water/acetonitrile (0.1% formic acid)Injection volume: 1 μL

TABLE 1 Gradient Time (min) Water Acetonitrile 0 95 5 0.1 95 5 2.1 5 953.0 STOP

Reverse-phase preparative HPLC purification was carried out using apreparative system manufactured by Waters Corp. under the followingconditions:

Column: YMC-Actus Triart C18 manufactured by YMC Co., Ltd., 20×50 mm, 5μm and YMC-Actus Triart C18 manufactured by YMC Co., Ltd., 20×10 mm, 5μm connected for useUV detection: 254 nmMS detection: ESI positiveColumn flow rate: 25 mL/minMobile phase: water/acetonitrile (0.1% formic acid)Injection volume: 0.1 to 0.5 mL

The meanings of abbreviations are as follows:

s: singletd: doublett: tripletq: quartetdd: double doubletdt: double triplettd: triple doublettt: triple tripletddd: double double doubletddt: double double tripletdtd: double triple doublettdd: triple double doubletm: multipletbr: broadbrs: broad singletDMSO-d₆: deuterated dimethyl sulfoxideCDCl₃: deuterated chloroformCD₃OD: deuterated methanolTHF: tetrahydrofuran

DMF: N,N-dimethylformamide DMA: N,N-dimethylacetamide

NMP: l-methyl-2-pyrrolidinoneDME: ethylene glycol dimethyl etherDMSO: dimethyl sulfoxideTFA: trifluoroacetic acidWSC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlorideHATU:(dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methaniminiumhexafluorophosphateDIPEA: diisopropylethylamineBoc: tert-butoxycarbonyl groupdppf: 1,1′-bis(diphenylphosphino)ferrocene

Reference Example 1 Synthesis of (R)-tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate

(Step 1) Synthesis of (S)-tert-butyl3-(methylsulfonyloxy)piperidine-1-carboxylate

(S)—N-Boc-3-piperidinol (20 g) was dissolved in toluene (100 mL). To thesolution, triethylamine (21 mL) and methanesulfonyl chloride (9.2 mL)were added at 0° C. The mixture was stirred under ice cooling for 1hour, and then, ethyl acetate and water were added thereto to separatean organic layer. The organic layer was washed with a saturated aqueoussolution of sodium bicarbonate, a saturated aqueous solution of ammoniumchloride, and water and then dried over anhydrous sodium sulfate, andthe solvent was distilled off under reduced pressure to obtain 26.8 g ofthe title compound as a colorless solid.

(Step 2) Synthesis of (R)-tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate

3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (14.6 g) synthesized by themethod described in the pamphlet of International Publication No. WO2007/126841, (S)-tert-butyl3-(methylsulfonyloxy)piperidine-1-carboxylate (25 g) obtained in (Step1), and potassium carbonate (69 g) were suspended in DMA (150 mL), andthe suspension was heated to 100° C. and stirred for 10 hours. After themixture was cooled to room temperature, water (300 mL) was addedthereto, and the precipitated solid was collected by filtration, washedwith water, and then dried to obtain 26.9 g of the title compound as ayellow solid.

Physical property value: m/z[M+H]+ 446.2

Reference Example 2 Synthesis of(R)-4-amino-1-(1-(tert-butyloxycarbonyl)piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicAcid

(R)-tert-Butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate(2 g) obtained in Reference Example 1, 2-diethylaminoethanol (3 mL), andPd(PPh₃)₂Cl₂ (158 mg) were dissolved in NMP (20 mL). After purging ofthe system with carbon monoxide, the solution was heated to 120° C. Thesolution was stirred for 1 hour and then cooled to room temperature.Methanol (10 mL) was added thereto, then a 5 N aqueous sodium hydroxidesolution (6 mL) was added thereto, and the mixture was stirred for 10minutes. After water was added, the aqueous layer was washed with ethylacetate and adjusted to pH 4 with hydrochloric acid, and theprecipitated solid was collected by filtration, washed with water, andthen dried to obtain 1.26 g of the title compound as a pale yellowsolid.

Physical property value: m/z[M+H]+ 363.1

Reference Example 3 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicAcid

To(R)-4-amino-1-(1-(tert-butyloxycarbonyl)piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 g) obtained in Reference Example 2, 4 N hydrochloricacid/1,4-dioxane (50 mL) was added, and the mixture was stirred for 1hour. Then, the solvent was removed with an evaporator. Chloroform (140mL) and triethylamine (25 mL) were added to the residue, and the mixturewas cooled in ice, followed by adding acryloyl chloride (2.23 mL). Themixture was stirred for 1.5 hours, and then, the solvent was removedwith an evaporator. To the residue, formic acid was added untiladjusting to pH 3.0, and the mixture was stirred for 2 hours. Then, theprecipitated solid was collected by filtration and dried under reducedpressure to obtain 8.93 g of the title compound as a white-brown solid.

Physical property value: m/z[M+H]+ 317.3

Reference Example 4 Synthesis of(R)-4-amino-1-(1-(tert-butyloxycarbonyl)pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic Acid

(Step 1) Synthesis of (S)-tert-butyl3-(methylsulfonyloxy)pyrrolidine-1-carboxylate

(S)-(−)-N-Boc-3-pyrrolidinol (935 mg) was dissolved in chloroform (15mL). To the solution, triethylamine (1.04 mL) and methanesulfonylchloride (467 μL) were added under ice cooling. The mixture was stirredat room temperature for 1.5 hours, and then, ethyl acetate and waterwere added thereto to separate an organic layer. The organic layer waswashed with a saturated aqueous solution of sodium bicarbonate, asaturated aqueous solution of ammonium chloride, and water and thendried over anhydrous sodium sulfate, and the solvent was distilled offunder reduced pressure to obtain 1.3 g of the title compound as acolorless oil.

Physical property value: m/z[M+H]+ 266.1

(Step 2) Synthesis of (R)-tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate

3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20.0 g) synthesized by themethod described in the pamphlet of International Publication No. WO2007/126841, (S)-tert-butyl 3-(methylsulfonyloxy)pyrrolidine-1-carboxylate (23 g) obtained in (Step 1), and potassiumcarbonate (32 g) were suspended in DMA (200 mL), and the suspension washeated to 85° C. and stirred for 3 hours. After the mixture was cooledto room temperature, water (400 mL) was added thereto, and theprecipitated solid was collected by filtration, washed with water, andthen dried to obtain 23.5 g of the title compound as a pale yellowsolid.

Physical property value: m/z[M+H]+ 431.0

(Step 3) Synthesis of(R)-4-amino-1-(1-(tert-butyloxycarbonyl)pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicAcid

(R)-tert-Butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate(2.0 g) obtained in (Step 2), 2-diethylaminoethanol (3.1 mL), andPd(PPh₃)₂Cl₂ (163 mg) were dissolved in NMP (20 mL). After the systemwas purged with carbon monoxide, the solution was heated to 120° C. Thesolution was stirred for 1 hour and then cooled to room temperature.Methanol (10 mL) was added thereto, then a 5 N aqueous sodium hydroxidesolution (6 mL) was added thereto, and the mixture was stirred for 10minutes. After water was added, the aqueous layer was washed withchloroform and adjusted to pH 4 with hydrochloric acid, and theprecipitated solid was collected by filtration, washed with water, andthen dried to obtain 1.35 g of the title compound as a pale yellowsolid.

Physical property value: m/z[M+H]+ 349.1

Reference Example 5 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicAcid

To(R)-4-amino-1-(1-(tert-butyloxycarbonyl)pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (1.0 g) obtained in Reference Example 4, 4 N hydrochloricacid/1,4-dioxane (5 mL) was added, and the mixture was stirred for 1hour. Then, the solvent was removed with an evaporator. Chloroform (28mL) and triethylamine (1.5 mL) were added to the residue, and themixture was cooled in ice, followed by adding acryloyl chloride (0.21mL). The mixture was stirred for 1.5 hours, and then, the solvent wasremoved with an evaporator. To the residue, formic acid was added untiladjusting to pH 3.0, and the mixture was stirred for 2 hours. Then, theprecipitated solid was collected by filtration and dried under reducedpressure to obtain 0.859 g of the title compound as a white-brown solid.

Physical property value: m/z[M+H]+ 303.3

Synthesis Example 1 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-bromo-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(2-bromo-5-methyl-4-nitrophenyl)acetic Acid

According to the method described in the pamphlet of InternationalPublication No. WO 2012/058134, NMP (10 mL) was added to diethylmalonate (2.4 g). Then, the solution was cooled in an ice bath, andsodium hydride (960 mg) was added thereto. The mixture was stirred at 0°C. for 30 minutes, and then, 1-bromo-2-fluoro-4-methyl-5-nitrobenzene(2.34 g) was added thereto. The mixture was stirred at room temperaturefor 1 hour, and then, ethyl acetate and water were added thereto toseparate an organic layer. The organic layer was washed with a saturatedaqueous solution of ammonium chloride and then dried over anhydroussodium sulfate, and the solvent was distilled off under reducedpressure. The residue was purified by silica gel column chromatography(hexane/ethyl acetate) to obtain 3.13 g of a colorless oil. Next,methanol (30 mL) and a 5 M aqueous sodium hydroxide solution (7.5 mL)were added to the obtained oil, and the mixture was heated to reflux for5 hours. After the solution was cooled to room temperature, the solventwas distilled off under reduced pressure, and a 2 M aqueous hydrochloricacid solution was added to the residue. The precipitated solid wascollected by filtration and dried to obtain 1.18 g of the title compoundas a yellow solid.

(Step 2) Synthesis of2-(2-bromo-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide

2-(2-Bromo-5-methyl-4-nitrophenyl)acetic acid (1.15 g) obtained in(Step 1) was dissolved in dichloromethane (20 mL). To the solution,oxalyl chloride (0.426 mL) and DMF (0.0653 mL) were added under icecooling, and then, the mixture was stirred at room temperature for 2hours. After concentration of the solution, toluene was added to theresidue, and the mixture was concentrated again. The obtained lightbrown oil was dissolved in chloroform (30 mL). To the solution, a 2.0 Msolution of dimethylamine in THF (10.5 mL) was added, and the mixturewas stirred for 1 hour. The solution was concentrated, and the residuewas dissolved in chloroform and washed with water. The obtained organiclayer was dried over anhydrous sodium sulfate, filtered, andconcentrated, and then, the residue was purified by silica gelchromatography (eluent: hexane-ethyl acetate) to obtain the titlecompound (1.15 g) as a yellow solid.

(Step 3) Synthesis of2-(4-amino-2-bromo-5-methylphenyl)-N,N-dimethylacetamide

To 2-(2-bromo-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide (300 mg)obtained in (Step 2), ethanol (3 mL) and water (3 mL) were added, andthen, ammonium chloride (300 mg) and iron powder (300 mg) were added.The solution was heated to 70° C., stirred for 2 hours, then cooled toroom temperature, and filtered through celite to remove iron. Ethylacetate was added to the filtrate for extraction. The extract was driedover anhydrous sodium sulfate, and the solvent was distilled off underreduced pressure. The obtained residue was purified by silica gelchromatography (hexane/ethyl acetate) to obtain 200 mg of the titlecompound as a yellow solid.

(Step 4) Synthesis of Synthesis Example Compound 1

(R)-1-(1-Acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (50 mg) obtained in Reference Example 3 and2-(4-amino-2-bromo-5-methylphenyl)-N,N-dimethylacetamide (64 mg)obtained in (Step 3) were dissolved by adding DMF (1 mL) and DIPEA(0.055 mL). To the solution, HATU (90 mg) was then added. The mixturewas stirred at room temperature for 1 hour, then purified byreverse-phase preparative HPLC purification (water/acetonitrile (0.1%formic acid)), and concentrated, and then, the residue was dissolved inchloroform, washed with a saturated aqueous solution of sodiumcarbonate, dried using anhydrous sodium sulfate, and concentrated. Then,the obtained residue was suspended in ethyl acetate-hexane, and thesuspension was filtered to obtain 58 mg of the title compound as anoff-white solid.

Synthesis Example 2 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(5-chloro-2-methyl-4-nitrophenyl)acetic Acid

According to (Step 1) of Synthesis Example 1, 952 mg of the titlecompound was obtained as a pale yellow solid by using1-chloro-5-fluoro-4-methyl-2-nitrobenzene (1.00 g) instead of1-bromo-2-fluoro-4-methyl-5-nitrobenzene.

(Step 2) Synthesis of2-(5-chloro-2-methyl-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 1, 1.03 g of the titlecompound was obtained as a pale yellow solid by using2-(5-chloro-2-methyl-4-nitrophenyl)acetic acid (922 mg) obtained in(Step 1) instead of 2-(2-bromo-5-methyl-4-nitrophenyl)acetic acid.

(Step 3) Synthesis of2-(4-amino-5-chloro-2-methylphenyl)-N,N-dimethylacetamide

According to (Step 3) of Synthesis Example 1, 720 mg of the titlecompound was obtained as a pale yellow solid by using2-(5-chloro-2-methyl-4-nitrophenyl)-N,N-dimethylacetamide (1.03 g)obtained in (Step 2) instead of2-(2-bromo-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 4) Synthesis of Synthesis Example Compound 2

According to (Step 4) of Synthesis Example 1,(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (66.5 mg) and2-(4-amino-5-chloro-2-methylphenyl)-N,N-dimethylacetamide (95.3 mg)obtained in (Step 3) were dissolved by adding DMSO (0.665 mL) and DIPEA(73.2 μL). To the solution, HATU (240 mg) was then added to obtain 56.2mg of the title compound as a white solid.

Synthesis Example 3 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(3-chloro-2-methyl-4-nitrophenyl)acetic Acid

According to (Step 1) of Synthesis Example 1, 1.23 g of the titlecompound was obtained as a pale yellow solid by using2-chloro-4-fluoro-3-methyl-1-nitrobenzene (1.00 g) instead of1-bromo-2-fluoro-4-methyl-5-nitrobenzene.

(Step 2) Synthesis of2-(3-chloro-2-methyl-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 1, 900 mg of the titlecompound was obtained as a pale yellow solid by using2-(3-chloro-2-methyl-4-nitrophenyl)acetic acid (1.23 g) obtained in(Step 1) instead of 2-(2-bromo-5-methyl-4-nitrophenyl)acetic acid.

(Step 3) Synthesis of2-(4-amino-3-chloro-2-methylphenyl)-N,N-dimethylacetamide

According to (Step 3) of Synthesis Example 1, 590 mg of the titlecompound was obtained as a white solid by using2-(3-chloro-2-methyl-4-nitrophenyl)-N,N-dimethylacetamide (900 mg)obtained in (Step 2) instead of2-(2-bromo-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 4) Synthesis of Synthesis Example Compound 3

According to (Step 4) of Synthesis Example 1,(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (66.5 mg) and2-(4-amino-3-chloro-2-methylphenyl)-N,N-dimethylacetamide (95.3 mg)obtained in (Step 3) were dissolved by adding DMSO (0.665 mL) and DIPEA(73.2 μL). To the solution, HATU (240 mg) was then added to obtain 78.2mg of the title compound as a white solid.

Synthesis Example 4 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of N,N-dimethyl-2-(3-methyl-4-nitrophenyl)acetamide

3-Methyl-4-nitrophenylacetic acid (50 mg) was dissolved by adding DMF(1.3 mL), DIEPA (0.13 mL), and a 2.0 M solution of dimethylamine in THF(0.38 mL). To the solution, HATU (145 mg) was then added. The mixturewas stirred at room temperature for 2 hours, and then, ethyl acetate andwater were added thereto to separate an organic layer. The organic layerwas washed with a saturated aqueous solution of sodium bicarbonate, asaturated aqueous solution of ammonium chloride, and water and thendried over anhydrous sodium sulfate, and the solvent was distilled offunder reduced pressure. The residue was purified by silica gelchromatography (hexane/ethyl acetate) to obtain 50 mg of the titlecompound as a colorless oil.

(Step 2) Synthesis of 2-(4-amino-3-methylphenyl)-N,N-dimethylacetamide

N,N-Dimethyl-2-(3-methyl-4-nitrophenyl)acetamide (50 mg) obtained in(Step 1) was dissolved in methanol (2.2 mL). To the solution, 10% Pd oncarbon (5 mg) was then added. The mixture was stirred overnight in ahydrogen atmosphere and then filtered through celite to remove Pd oncarbon. The solvent was distilled off under reduced pressure to obtain40 mg of the title compound as a colorless oil.

(Step 3) Synthesis of Synthesis Example Compound 4

(R)-1-(1-Acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) obtained in Reference Example 3 and2-(4-amino-3-methylphenyl)-N,N-dimethylacetamide (9.2 mg) obtained in(Step 2) were dissolved by adding DMF (0.2 mL) and DIPEA (20 μL). To thesolution, HATU (18 mg) was then added. The mixture was stirred at roomtemperature for 1 hour. Then, DMSO (1 mL) was added thereto, and themixture was purified by reverse-phase preparative HPLC purification(water/acetonitrile (0.1% formic acid)) to obtain 3.4 mg of the titlecompound as a white solid.

Synthesis Example 5 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of N,N-dimethyl-2-(4-nitronaphthalen-1-yl)acetamide

1-Naphthylacetic acid (500 mg) was dissolved in DMF. To the solution, a2.0 M solution of diethylamine in THF (2.7 mL), DIPEA (1.4 mL), and HATU(1.3 g) were then added. The mixture was stirred at room temperature for2 hours, and then, ethyl acetate and water were added thereto toseparate an organic layer. The organic layer was washed with a saturatedaqueous solution of sodium bicarbonate and water and then dried overanhydrous sodium sulfate, and the solvent was distilled off underreduced pressure. Sulfuric acid (5 mL) was added to the obtainedresidue, and the solution was cooled to 0° C. Then, fuming nitric acid(I mL) was added dropwise thereto The mixture was stirred at 0° C. for30 minutes, and then, the solution was added to an aqueous sodiumbicarbonate solution and neutralized, followed by extraction with ethylacetate. The extract was dried over anhydrous sodium sulfate, and thesolvent was distilled off under reduced pressure. The obtained residuewas purified by silica gel chromatography (hexane/ethyl acetate) toobtain 310 mg of the title compound as a yellow solid.

(Step 2) Synthesis of 2-(4-aminonaphthalen-1-yl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 4, 18 mg of the titlecompound was obtained as a colorless oil by usingN,N-dimethyl-2-(4-nitronaphthalen-1-yl)acetamide (20 mg).

(Step 3) Synthesis of Synthesis Example Compound 5

According to (Step 3) of Synthesis Example 4, 8.9 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-aminonaphthalen-1-yl)-N,N-dimethylacetamide (11mg) obtained in (Step 2)

Synthesis Example 6 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 890 mg of the titlecompound was obtained as a brown oil by using2-(3-chloro-4-nitrophenyl)acetic acid (980 mg) instead of3-methyl-4-nitrophenylacetic acid.

(Step 2) Synthesis of 2-(4-amino-3-chlorophenyl)-N,N-dimethylacetamide

To 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide (1.4 g) obtained in(Step 1), ethanol (14 mL) and water (14 mL) were added, and then,ammonium chloride (1.4 g) and iron powder (1.4 g) were added. Thesolution was heated to 70° C., stirred for 2 hours, then cooled to roomtemperature, and filtered through celite to remove iron. Ethyl acetatewas added to the filtrate for extraction. The extract was dried overanhydrous sodium sulfate, and the solvent was distilled off underreduced pressure. The obtained residue was purified by silica gelchromatography (hexane/ethyl acetate) to obtain 1.1 g of the titlecompound as a yellow solid.

(Step 3) Synthesis of Synthesis Example Compound 6

According to (Step 3) of Synthesis Example 4, 6.0 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (25 mg) and 2-(4-amino-3-chlorophenyl)-N,N-dimethylacetamide (25mg) obtained in (Step 2).

Synthesis Example 7 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(3-methoxy-4-nitrophenyl)-N,N-dimethylacetamide

According to the synthesis method described in European Journal ofMedicinal Chemistry, 46 (1), 285-296; 2010, 480 mg of the title compoundwas obtained as a yellow oil by using 3-methoxy-4-nitrobenzaldehyde (510mg) instead of 4-nitrobenzaldehyde.

(Step 2) Synthesis of 2-(4-amino-3-methoxyphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 450 mg of the titlecompound was obtained as a yellow oil by using2-(3-methoxy-4-nitrophenyl)-N,N-dimethylacetamide (480 mg) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 7

According to (Step 3) of Synthesis Example 4, 18 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (30 mg) and 2-(4-amino-3-methoxyphenyl)-N,N-dimethylacetamide (29mg) obtained in (Step 2).

Synthesis Example 8 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(2-chloro-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 220 mg of the titlecompound was obtained as a yellow solid by using2-(2-chloro-4-nitrophenyl)acetic acid (220 mg) instead of3-methyl-4-nitrophenylacetic acid.

(Step 2) Synthesis of 2-(4-amino-2-chlorophenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 180 mg of the titlecompound was obtained as a yellow oil by using2-(2-chloro-4-nitrophenyl)-N,N-dimethylacetamide (220 mg) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 8

According to (Step 3) of Synthesis Example 4, 13.5 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-2-chlorophenyl)-N,N-dimethylacetamide (10mg) obtained in (Step 2).

Synthesis Example 9 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of2-(2-chloro-5-methoxy-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 388 mg of the titlecompound was obtained as a yellow solid by using2-(2-chloro-5-methoxy-4-nitrophenyl)acetic acid (350 mg) instead of3-methyl-4-nitrophenylacetic acid.

(Step 2) Synthesis of2-(4-amino-2-chloro-5-methoxyphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 340 mg of the titlecompound was obtained as a yellow oil by using2-(2-chloro-5-methoxy-4-nitrophenyl)-N,N-dimethylacetamide (388 mg)instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 9

According to (Step 3) of Synthesis Example 4, 17.5 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and2-(4-amino-2-chloro-5-methoxyphenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 2).

Synthesis Example 10 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-5-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(2-fluoro-5-methyl-4-nitrophenyl) acetic Acid

According to the method described in the pamphlet of InternationalPublication No. WO 2012/058134, NMP (30 mL) was added to diethylmalonate (1.0 g). Then, the solution was cooled in an ice bath, andsodium hydride (380 mg) was added thereto. The mixture was stirred at 0°C. for 30 minutes, and then, 1,2-difluoro-4-methyl-5-nitrobenzene (690mg) was added thereto. The mixture was stirred at room temperature for 1hour, and then, ethyl acetate and water were added thereto to separatean organic layer. The organic layer was washed with a saturated aqueoussolution of ammonium chloride and then dried over anhydrous sodiumsulfate, and the solvent was distilled off under reduced pressure. Theresidue was purified by silica gel chromatography (hexane/ethyl acetate)to obtain 1.20 g of a colorless oil. Next, methanol (10 mL) and a 5 Maqueous sodium hydroxide solution (2.5 mL) were added to the obtainedoil, and the mixture was heated to reflux for 5 hours. After thesolution was cooled to room temperature, the solvent was distilled offunder reduced pressure, and a 2 M aqueous hydrochloric acid solution wasadded to the residue. The precipitated solid was collected by filtrationand dried to obtain 610 mg of the title compound as a yellow solid.

(Step 2) Synthesis of2-(2-fluoro-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 600 mg of the titlecompound was obtained as a yellow oil by using2-(2-fluoro-5-methyl-4-nitrophenyl)acetic acid (610 mg) instead of3-methyl-4-nitrophenylacetic acid.

(Step 3) Synthesis of2-(4-amino-2-fluoro-5-methylphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 540 mg of the titlecompound was obtained as a yellow oil by using2-(2-fluoro-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide (600 mg)instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 4) Synthesis of Synthesis Example Compound 10

According to (Step 3) of Synthesis Example 4, 7.3 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and2-(4-amino-2-fluoro-5-methylphenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 3).

Synthesis Example 11 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(2-chloro-3-methoxy-4-nitrophenyl)acetic Acid

According to (Step 1) of Synthesis Example 10, 710 mg of the titlecompound was obtained as a yellow solid by using2-chloro-1-fluoro-3-methoxy-4-nitrobenzene (800 mg) instead of1,2-difluoro-4-methyl-5-nitrobenzene.

(Step 2) Synthesis of2-(2-chloro-3-methoxy-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 110 mg of the titlecompound was obtained as a yellow oil by using2-(2-chloro-3-methoxy-4-nitrophenyl)acetic acid (100 mg) obtained in(Step 1) instead of 3-methyl-4-nitrophenylacetic acid.

(Step 3) Synthesis of2-(4-amino-2-chloro-3-methoxyphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 95 mg of the titlecompound was obtained as a yellow oil by using2-(2-chloro-3-methoxy-4-nitrophenyl)-N,N-dimethylacetamide (110 mg)obtained in (Step 2) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 4) Synthesis of Synthesis Example Compound 11

According to (Step 3) of Synthesis Example 4, 6.9 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and2-(4-amino-2-chloro-3-methoxyphenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 3).

Synthesis Example 12 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(2-fluoro-3-methyl-4-nitrophenyl)acetic Acid

According to (Step 1) of Synthesis Example 10, 800 mg of the titlecompound was obtained as a yellow solid by using1,2-difluoro-3-methyl-4-nitrobenzene (560 mg) instead of1,2-difluoro-4-methyl-5-nitrobenzene.

(Step 2) Synthesis of2-(2-fluoro-3-methyl-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 290 mg of the titlecompound was obtained as a yellow oil by using2-(2-fluoro-3-methyl-4-nitrophenyl)acetic acid (260 mg) obtained in(Step 1) instead of 3-methyl-4-nitrophenylacetic acid.

(Step 3) Synthesis of2-(4-amino-2-fluoro-3-methylphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 230 mg of the titlecompound was obtained as a yellow oil by using2-(2-fluoro-3-methyl-4-nitrophenyl)-N,N-dimethylacetamide (290 mg)obtained in (Step 2) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 4) Synthesis of Synthesis Example Compound 12

According to (Step 3) of Synthesis Example 4, 8.2 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and2-(4-amino-2-fluoro-3-methylphenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 3).

Synthesis Example 13 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(2,3-dimethyl-4-nitrophenyl)acetic Acid

According to (Step 1) of Synthesis Example 10, 1.12 g of the titlecompound was obtained as a yellow solid by using1-fluoro-2,3-dimethyl-4-nitrobenzene (1.2 g) instead of1,2-difluoro-4-methyl-5-nitrobenzene.

(Step 2) Synthesis of2-(2,3-dimethyl-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 1.30 g of the titlecompound was obtained as a yellow oil by using2-(2,3-dimethyl-4-nitrophenyl)acetic acid (1.12 g) obtained in (Step 1)instead of 3-methyl-4-nitrophenylacetic acid.

(Step 3) Synthesis of2-(4-amino-2,3-dimethylphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 1.03 g of the titlecompound was obtained as a yellow oil by using2-(2,3-dimethyl-4-nitrophenyl)-N,N-dimethylacetamide (1.26 g) obtainedin (Step 2) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 4) Synthesis of Synthesis Example Compound 13

According to (Step 3) of Synthesis Example 4, 48 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (40 mg) and 2-(2,3-dimethyl-4-nitrophenyl)-N,N-dimethylacetamide(31 mg) obtained in (Step 3).

Synthesis Example 14 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(2-fluoro-3-methoxy-4-nitrophenyl)acetic Acid

According to (Step 1) of Synthesis Example 10, 1.10 g of the titlecompound was obtained as a yellow solid by using1,2-difluoro-3-methoxy-4-nitrobenzene (1.0 g) instead of1,2-difluoro-4-methyl-5-nitrobenzene.

(Step 2) Synthesis of2-(2-fluoro-3-methoxy-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 120 mg of the titlecompound was obtained as a yellow oil by using2-(2-fluoro-3-methoxy-4-nitrophenyl)acetic acid (100 mg) obtained in(Step 1) instead of 3-methyl-4-nitrophenylacetic acid.

(Step 3) Synthesis of2-(4-amino-2-fluoro-3-methoxyphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 100 mg of the titlecompound was obtained as a yellow oil by using2-(2-fluoro-3-methoxy-4-nitrophenyl)-N,N-dimethylacetamide (120 mg)obtained in (Step 2) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 4) Synthesis of Synthesis Example Compound 14

According to (Step 3) of Synthesis Example 4, 4.9 mg of the titlecompound was obtained as a yellow solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and2-(4-amino-2-fluoro-3-methoxyphenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 3).

Synthesis Example 15 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-(difluoromethoxy)-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of2-(3-(difluoromethoxy)-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 450 mg of2-(3-hydroxy-4-nitrophenyl)-N,N-dimethylacetamide was obtained as ayellow oil by using 2-(3-hydroxy-4-nitrophenyl)acetic acid (420 mg)instead of 3-methyl-4-nitrophenylacetic acid. Next, DMF (4.5 mL) andpotassium carbonate (250 mg) were added to the obtained2-(3-hydroxy-4-nitrophenyl)-N,N-dimethylacetamide (200 mg), and sodium2-chloro-2,2-difluoroacetate (270 mg) was added to the suspension. Themixture was stirred at room temperature for 1 hour, and then, ethylacetate and water were added thereto to separate an organic layer. Theorganic layer was washed with a saturated aqueous solution of ammoniumchloride and then dried over anhydrous sodium sulfate, and the solventwas distilled off under reduced pressure. The residue was purified bysilica gel chromatography (hexane/ethyl acetate) to obtain 155 mg of thetitle compound as a yellow solid.

(Step 2) Synthesis of2-(4-amino-3-(difluoromethoxy)phenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 120 mg of the titlecompound was obtained as a yellow oil by using2-(3-(difluoromethoxy)-4-nitrophenyl)-N,N-dimethylacetamide (155 mg)obtained in (Step 1) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 15

According to (Step 3) of Synthesis Example 4, 13.5 mg of the titlecompound was obtained as a yellow solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and2-(4-amino-3-(difluoromethoxy)phenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 2).

Synthesis Example 16 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-(fluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of2-(3-(fluoromethoxy)-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 450 mg of2-(3-hydroxy-4-nitrophenyl)-N,N-dimethylacetamide was obtained as ayellow oil by using 2-(3-hydroxy-4-nitrophenyl)acetic acid (420 mg)instead of 3-methyl-4-nitrophenylacetic acid. Next, DMF (4.5 mL) andpotassium carbonate (370 mg) were added to the obtained2-(3-hydroxy-4-nitrophenyl)-N,N-dimethylacetamide (200 mg), andbromo-fluoromethane (200 mg) was added to the suspension. The mixturewas stirred at room temperature for 1 hour, and then, ethyl acetate andwater were added thereto to separate an organic layer. The organic layerwas washed with a saturated aqueous solution of ammonium chloride andthen dried over anhydrous sodium sulfate, and the solvent was distilledoff under reduced pressure. The residue was purified by silica gelchromatography (hexane/ethyl acetate) to obtain 145 mg of the titlecompound as a yellow solid.

(Step 2) Synthesis of2-(4-amino-3-(fluoromethoxy)phenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 110 mg of the titlecompound was obtained as a yellow oil by using2-(3-(fluoromethoxy)-4-nitrophenyl)-N,N-dimethylacetamide (145 mg)obtained in (Step 1) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 16

According to (Step 3) of Synthesis Example 4, 14.5 mg of the titlecompound was obtained as a yellow solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and2-(4-amino-3-(fluoromethoxy)phenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 2).

Synthesis Example 17 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-bromo-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(3-bromo-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 440 mg of the titlecompound was obtained as a yellow oil by using2-(3-bromo-4-nitrophenyl)acetic acid (430 mg) instead of3-methyl-4-nitrophenylacetic acid.

(Step 2) Synthesis of 2-(4-amino-3-bromophenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 80 mg of the titlecompound was obtained as a yellow oil by using2-(3-bromo-4-nitrophenyl)-N,N-dimethylacetamide (100 mg) obtained in(Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 17

According to (Step 3) of Synthesis Example 4, 2.10 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-3-bromophenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 2).

Synthesis Example 18 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(5-chloro-2-fluoro-4-nitrophenyl)acetic Acid

According to (Step 1) of Synthesis Example 10, 275 mg of the titlecompound was obtained as a yellow solid by using1-chloro-4,5-difluoro-2-nitrobenzene (500 mg) instead of1,2-difluoro-4-methyl-5-nitrobenzene.

(Step 2) Synthesis of2-(5-chloro-2-fluoro-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 205 mg of the titlecompound was obtained as a yellow oil by using2-(5-chloro-2-fluoro-4-nitrophenyl)acetic acid (234 mg) obtained in(Step 1) instead of 3-methyl-4-nitrophenylacetic acid.

(Step 3) Synthesis of2-(4-amino-5-chloro-2-fluorophenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 150 mg of the titlecompound was obtained as a yellow solid by using2-(5-chloro-2-fluoro-4-nitrophenyl)-N,N-dimethylacetamide (205 mg)obtained in (Step 2) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 4) Synthesis of Synthesis Example Compound 18

According to (Step 3) of Synthesis Example 4,(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and2-(4-amino-5-chloro-2-fluorophenyl)-N,N-dimethylacetamide (20 mg)obtained in (Step 3) were dissolved by adding DMSO (0.2 mL) and DIPEA(20 μL). To the solution, HATU (50 mg) was then added to obtain 2.34 mgof the title compound as a white solid.

Synthesis Example 19 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 1-chloro-2-fluoro-4-methyl-5-nitrobenzene

1-Chloro-2-fluoro-4-methylbenzene (1.00 g) was dissolved in TFA (10 mL).The solution was cooled to 0° C., and then, fuming nitric acid (5.0 mL)was added dropwise thereto. The mixture was stirred at 0° C. for 30minutes, and then, the solution was added to an aqueous sodiumbicarbonate solution and neutralized, followed by extraction withchloroform. The extract was dried over anhydrous sodium sulfate, and thesolvent was distilled off under reduced pressure. The obtained residuewas purified by silica gel chromatography (hexane/ethyl acetate) toobtain 1.31 g of the title compound as a white solid.

(Step 2) Synthesis of 2-(2-chloro-5-methyl-4-nitrophenyl)acetic Acid

According to (Step 1) of Synthesis Example 1, 440 mg of the titlecompound was obtained as a yellow solid by using1-chloro-2-fluoro-4-methyl-5-nitrobenzene (500 mg) obtained in (Step 1)instead of 1-bromo-2-fluoro-4-methyl-5-nitrobenzene.

(Step 3) Synthesis of2-(2-chloro-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 1, 149 mg of the titlecompound was obtained as a pale yellow solid by using2-(2-chloro-5-methyl-4-nitrophenyl)acetic acid (230 mg) obtained in(Step 2) instead of 2-(2-bromo-5-methyl-4-nitrophenyl)acetic acid.

(Step 4) Synthesis of2-(4-amino-2-chloro-5-methylphenyl)-N,N-dimethylacetamide

According to (Step 3) of Synthesis Example 1, 170 mg of the titlecompound was obtained as a white solid by using2-(2-chloro-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide (149 mg)obtained in (Step 3) instead of2-(2-bromo-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 5) Synthesis of Synthesis Example Compound 19

According to (Step 4) of Synthesis Example 1,(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and2-(4-amino-2-chloro-5-methylphenyl)-N,N-dimethylacetamide (20 mg)obtained in (Step 4) were dissolved by adding DMSO (0.2 mL) and DIPEA(20 μL). To the solution, HATU (50 mg) was then added to obtain 3.39 mgof the title compound as a white solid.

Synthesis Example 20 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2,5-dichloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 1,4-dichloro-2-fluoro-5-nitrobenzene

According to (Step 1) of Synthesis Example 19,1,4-dichloro-2-fluorobenzene (1.00 g) was dissolved instead of1-chloro-2-fluoro-4-methylbenzene in TFA (10 mL). To the solution,fuming nitric acid (5.0 mL) was added dropwise. The mixture was stirredat room temperature for 3 hours, and then, the solution was added to anaqueous sodium bicarbonate solution. The precipitated solid wascollected by filtration and dried to obtain 1.30 g of the title compoundas a white solid.

(Step 2) Synthesis of 2-(2,5-dichloro-4-nitrophenyl)acetic Acid

According to (Step 1) of Synthesis Example 1, 423 mg of the titlecompound was obtained as a yellow solid by using1,4-dichloro-2-fluoro-5-nitrobenzene (500 mg) obtained in (Step 1)instead of 1-bromo-2-fluoro-4-methyl-5-nitrobenzene.

(Step 3) Synthesis of2-(2,5-dichloro-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 1, 150 mg of the titlecompound was obtained as a pale yellow solid by using2-(2,5-dichloro-4-nitrophenyl)acetic acid (250 mg) obtained in (Step 2)instead of 2-(2-bromo-5-methyl-4-nitrophenyl)acetic acid.

(Step 4) Synthesis of2-(4-amino-2,5-dichlorophenyl)-N,N-dimethylacetamide

According to (Step 3) of Synthesis Example 1, 160 mg of the titlecompound was obtained as a white solid by using2-(2,5-dichloro-4-nitrophenyl)-N,N-dimethylacetamide (150 mg) obtainedin (Step 3) instead of2-(2-bromo-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 5) Synthesis of Synthesis Example Compound 20

According to (Step 4) of Synthesis Example 1,(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-2,5-dichlorophenyl)-N,N-dimethylacetamide(20 mg) obtained in (Step 4) were dissolved by adding DMSO (0.2 mL) andDIPEA (20 μL). To the solution, HATU (50 mg) was then added to obtain0.89 mg of the title compound as a white solid.

Synthesis Example 21 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)phenyldimethylcarbamate (Step 1) Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

(R)-1-(1-Acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (30 mg) synthesized in Reference Example 3 was dissolved by addingDMF (4.5 mL), 4-aminophenol (17 mg), and DIPEA (50 μL). To the solution,HATU (55 mg) was then added. The mixture was stirred at room temperaturefor 1 hour, and then, ethyl acetate and water were added thereto toseparate an organic layer. The organic layer was dried over anhydroussodium sulfate, and the solvent was distilled off under reducedpressure. The residue was purified by silica gel chromatography(chloroform/methanol) to obtain 25 mg of the title compound as acolorless oil.

(Step 2) Synthesis of Synthesis Example Compound 21

To(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(10 mg) obtained in (Step 1), potassium carbonate (10 mg) andacetonitrile (250 μL) were added to prepare a suspension. Then,N,N-dimethylcarbamoyl chloride (8 mg) was added thereto. The mixture wasstirred at 60° C. for 3 hours, and then, chloroform and water were addedthereto to separate an organic layer. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off underreduced pressure. The residue was purified by reverse-phase preparativeHPLC purification (water/acetonitrile (0.1% formic acid)) to obtain 2.62mg of the title compound as a white solid.

Synthesis Example 22 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-cyclopropyl-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-cyclopropyl-4-fluoro-1-nitrobenzene

2-Bromo-4-fluoro-1-nitrobenzene (1.22 g), potassium phosphate (4.12 g),and cyclopropylboronic acid were suspended in toluene (15 mL). To thesuspension, water (0.75 mL), palladium acetate (0.062 mg), andtricyclohexylphosphine (155 mg) were added, and the mixture was heatedto reflux in a nitrogen atmosphere and stirred for 4 hours. The mixturewas allowed to cool, and then, water was added thereto, followed byextraction with ethyl acetate. The obtained organic layer was washedwith water and subsequently washed with saturated saline. The obtainedorganic layer was dried using anhydrous sodium sulfate, filtered, andconcentrated, and then, the residue was purified by silica gelchromatography to obtain the title compound (895 mg) as a light brownoil.

(Step 2) Synthesis of 2-(3-cyclopropyl-4-nitrophenyl)acetic Acid

The title compound (845 mg) was obtained as a light orange solid in thesame way as in the approach of (Step 1) of Synthesis Example 10 by using2-cyclopropyl-4-fluoro-1-nitrobenzene (895 mg) instead of1,2-difluoro-4-methyl-5-nitrobenzene as a starting material.

(Step 3) Synthesis of2-(3-cyclopropyl-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, the title compound (445mg) was obtained by using 2-(3-cyclopropyl-4-nitrophenyl)acetic acid(895 mg) instead of 3-methyl-4-nitrophenylacetic acid.

(Step 4) Synthesis of2-(4-amino-3-cyclopropylphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, the title compound (225mg) was obtained as a light brown oil by using2-(3-cyclopropyl-4-nitrophenyl)-N,N-dimethylacetamide (445 mg) insteadof 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 5) Synthesis of Synthesis Example Compound 22

According to (Step 4) of Synthesis Example 1,(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (50 mg) obtained in Reference Example 3 and2-(4-amino-3-cyclopropylphenyl)-N,N-dimethylacetamide (51 mg) obtainedin (Step 4) were dissolved by adding DMF (1 mL) and DIPEA (0.055 mL). Tothe solution, HATU (90 mg) was then added to obtain 52 mg of the titlecompound as a colorless solid.

Synthesis Example 23 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-cyano-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(4-amino-3-bromophenyl)-N,N-dimethylacetamide

2-(4-Aminophenyl)acetic acid (1.03 g) was suspended in THF (40 mL). Tothe suspension, N-bromosuccinimide (1.27 g) was then added at roomtemperature, and then, the mixture was stirred for 1 hour. The solutionwas concentrated. To the residue, a 2.0 M solution of dimethylamine inTHF (17 mL) was added, and subsequently, HATU (3.89 g) was added at roomtemperature. The mixture was stirred at room temperature. Then, thesolution was concentrated, and the residue was diluted with ethylacetate. The ethyl acetate solution was washed with water, then washedwith a 1 N aqueous sodium hydroxide solution, and subsequently washedwith saturated saline. The obtained organic layer was dried overanhydrous sodium sulfate, filtered, and concentrated, and the residuewas purified by silica gel chromatography (eluent: hexane-ethyl acetate)to obtain the title compound (1.07 g) as a pale yellow oil.

(Step 2) Synthesis of 2-(4-amino-3-cyanophenyl)-N,N-dimethylacetamide

2-(4-Amino-3-bromophenyl)-N,N-dimethylacetamide (1.07 g) obtained in(Step 1) was dissolved in DMF (10 mL). After purging with nitrogen,tetrakis(triphenylphosphine)palladium (481 mg) and zinc cyanide (977 mg)were added to the solution, and the mixture was stirred at 120° C. for16 hours. The mixture was allowed to cool to room temperature and thendiluted with ethyl acetate. A 28% aqueous ammonium solution was addedthereto, and the mixture was stirred for 10 minutes. After extractionwith ethyl acetate, the extract was washed with a 28% aqueous ammoniumsolution again, then washed with water, and washed with saturatedsaline. The obtained organic layer was dried over anhydrous sodiumsulfate, filtered, and concentrated, and the residue was purified bysilica gel chromatography (eluent: hexane-ethyl acetate) to obtain thetitle compound (360 mg) as a pale yellow solid.

(Step 3) Synthesis of Synthesis Example Compound 23

According to (Step 4) of Synthesis Example 1,(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (50 mg) obtained in Reference Example 3 and2-(4-amino-3-cyanophenyl)-N,N-dimethylacetamide (48 mg) obtained in(Step 2) were dissolved by adding DMF (1 mL) and DIPEA (0.055 mL). Tothe solution, HATU (90 mg) was then added to obtain 12 mg of the titlecompound as a colorless solid.

Synthesis Example 24 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-cyano-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of2-(2-cyano-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide

2-(2-Bromo-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide (500 mg)obtained in (Step 2) of Synthesis Example 1 was dissolved in DMF (5 mL).After purging with nitrogen, tetrakis(triphenylphosphine)palladium (191mg) and zinc cyanide (390 mg) were added to the solution, and themixture was stirred at 120° C. for 16 hours. The mixture was allowed tocool to room temperature and then diluted with ethyl acetate. A 28%aqueous ammonium solution was added thereto, and the mixture was stirredfor 10 minutes. After extraction with ethyl acetate, the extract waswashed with a 28% aqueous ammonium solution again, then washed withwater, and washed with saturated saline. The obtained organic layer wasdried over anhydrous sodium sulfate, filtered, and concentrated, and theresidue was purified by silica gel chromatography (eluent: hexane-ethylacetate) to obtain the title compound (209 mg) as a pale yellow solid.

(Step 2) Synthesis of2-(4-amino-2-cyano-5-methylphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 4, the title compound (143mg) was obtained as a colorless solid by using2-(2-cyano-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide (200 mg)instead of N,N-dimethyl-2-(3-methyl-4-nitrophenyl)acetamide.

(Step 3) Synthesis of Synthesis Example Compound 24

According to (Step 4) of Synthesis Example 1,(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (50 mg) obtained in Reference Example 3 and2-(4-amino-2-cyano-5-methylphenyl)-N,N-dimethylacetamide (51 mg)obtained in (Step 2) were dissolved by adding DMF (1 mL) and DIPEA(0.055 mL). To the solution, HATU (90 mg) was then added to obtain 57 mgof the title compound as an off-white solid.

Synthesis Example 25 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-oxoethyl)-2-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(4-amino-3-fluorophenyl)-N,N-dimethylacetamide

2-(4-Amino-3-fluorophenyl)acetic acid (400 mg) was suspended in a 2.0 Msolution of dimethylamine in THF (7 mL). To the suspension, WSC (0.58 g)was then added at room temperature, and the mixture was stirred at roomtemperature for 1 hour. A 2.0 M solution of dimethylamine in THF (7 mL)was added thereto, subsequently HATU (1.16 g) was added thereto, and themixture was stirred for 30 minutes. After concentration of the solution,the residue was purified by silica gel chromatography (eluent:hexane-ethyl acetate) to obtain the title compound (300 mg) as a lightbrown oil.

(Step 2) Synthesis of Synthesis Example Compound 25

According to (Step 4) of Synthesis Example 1,(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (50 mg) obtained in Reference Example 3 and2-(4-amino-3-fluorophenyl)-N,N-dimethylacetamide (46 mg) obtained in(Step 1) were dissolved by adding DMF (1 mL) and DIPEA (0.055 mL). Tothe solution, HATU (90 mg) was then added to obtain 8.1 mg of the titlecompound as a colorless solid.

Synthesis Example 26 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-3-methylphenyldimethylcarbamate (Step 1) Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-hydroxy-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

(R)-1-(1-Acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (30 mg) synthesized in Reference Example 3 was dissolved by addingDMF (4.5 mL), 4-amino-3-methylphenol (17 mg), and DTPEA (50 μL). To thesolution, HATU (55 mg) was then added. The mixture was stirred at roomtemperature for 1 hour, and then, ethyl acetate and water were addedthereto to separate an organic layer. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off underreduced pressure. The residue was purified by silica gel chromatography(chloroform/methanol) to obtain 25 mg of the title compound as acolorless oil.

(Step 2) Synthesis of Synthesis Example Compound 26

To(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-hydroxy-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(10 mg) obtained in (Step 1), potassium carbonate (10 mg) andacetonitrile (250 μL) were added to prepare a suspension. Then,N,N-dimethylcarbamoyl chloride (8 mg) was added thereto. The mixture wasstirred at 60° C. for 3 hours, and then, chloroform and water were addedthereto to separate an organic layer. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off underreduced pressure. The residue was purified by reverse-phase preparativeHPLC purification (water/acetonitrile (0.1% formic acid)) to obtain 2.62mg of the title compound as a white solid.

Synthesis Example 27 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-3-chlorophenyldimethylcarbamate (Step 1) Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

According to (Step 1) of Synthesis Example 26, 41 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (30 mg) and 4-amino-3-chlorophenol (25 mg).

(Step 2) Synthesis of Synthesis Example Compound 27

According to (Step 2) of Synthesis Example 26, 8.1 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(10 mg) obtained in (Step 1) instead of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-hydroxy-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.

Synthesis Example 28 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of N,N-dimethyl-2-(4-nitrophenyl)acetamide

According to (Step 1) of Synthesis Example 4, 60 mg of the titlecompound was obtained as a yellow oil by using 2-(4-nitrophenyl)aceticacid (50 mg) instead of 3-methyl-4-nitrophenylacetic acid.

(Step 2) Synthesis of 2-(4-aminophenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 50 mg of the titlecompound was obtained as a yellow oil by usingN,N-dimethyl-2-(4-nitrophenyl)acetamide (60 mg) obtained in (Step 1)instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 28

According to (Step 3) of Synthesis Example 4, 5.22 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (7.5 mg) and 2-(4-aminophenyl)-N,N-dimethylacetamide (7.5 mg)obtained in (Step 2).

Synthesis Example 29 Synthesis of(R)-4-(l-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-2-chlorophenyldimethylcarbamate (Step 1) Synthesis of 2-chloro-4-nitrophenyldimethylcarbamate

To 2-chloro-4-nitrophenol (100 mg), potassium carbonate (160 mg) andacetonitrile (3.0 mL) were added, and N,N-dimethylcarbamoyl chloride(100 mg) was added to the suspension. The mixture was stirred at 60° C.for 2 hours, and then, ethyl acetate and water were added thereto toseparate an organic layer. The organic layer was dried over anhydroussodium sulfate, and the solvent was distilled off under reducedpressure. The residue was purified by silica gel chromatography(hexane/ethyl acetate) to obtain 120 mg of the title compound as acolorless oil.

(Step 2) Synthesis of 4-amino-2-chlorophenyl dimethylcarbamate

According to (Step 2) of Synthesis Example 6, 95 mg of the titlecompound was obtained as a yellow oil by using 2-chloro-4-nitrophenyldimethylcarbamate (120 mg) obtained in (Step 1) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 29

According to (Step 3) of Synthesis Example 4, 4.27 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 4-amino-2-chlorophenyl dimethylcarbamate (10 mg)obtained in (Step 2).

Synthesis Example 30 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-2-methylphenyldimethylcarbamate (Step 1) Synthesis of Synthesis Example Compound 30

(R)-1-(1-Acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) synthesized in Reference Example 3 was dissolved by addingDMF (0.2 mL), 4-amino-2-methylphenol (7 mg), and DIPEA (20 μL). To thesolution, HATU (18 mg) was then added. The mixture was stirred at roomtemperature for 1 hour, and then, the solution was purified by silicagel chromatography (chloroform/methanol) to obtain 10 mg of a whitesolid compound. Next, potassium carbonate (10 mg) and acetonitrile (250μL) were added to the obtained compound (10 mg) to prepare a suspension.Then, N,N-dimethylcarbamoyl chloride (12 mg) was added thereto. Themixture was stirred at 60° C. for 3 hours, and then, chloroform andwater were added thereto to separate an organic layer. The organic layerwas dried over anhydrous sodium sulfate, and the solvent was distilledoff under reduced pressure. The residue was purified by reverse-phasepreparative HPLC purification (water/acetonitrile (0.1% formic acid)) toobtain 3.27 mg of the title compound as a white solid.

Synthesis Example 31 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-3-fluorophenyldimethylcarbamate (Step 1) Synthesis of Synthesis Example Compound 31

According to Synthesis Example 30, 2.47 mg of the title compound wasobtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 4-amino-3-fluorophenol (7 mg).

Synthesis Example 32 Synthesis of(R)-4-(l-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-2,3-dimethylphenyldimethylcarbamate (Step 1) Synthesis of Synthesis Example Compound 32

According to Synthesis Example 30, 2.67 mg of the title compound wasobtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 4-amino-2,3-dimethylphenol (7 mg).

Synthesis Example 33 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)naphthalen-1-yldimethylcarbamate (Step 1) Synthesis of Synthesis Example Compound 33

According to Synthesis Example 30, 2.60 mg of the title compound wasobtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 4-aminonaphthol (10 mg).

Synthesis Example 34 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-2-fluorophenyldimethylcarbamate (Step 1) Synthesis of Synthesis Example Compound 34

According to Synthesis Example 30, 1.92 mg of the title compound wasobtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 4-amino-2-fluorophenol (7 mg).

Synthesis Example 35 Synthesis of(R)-4-(l-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-2,3-difluorophenyldimethylcarbamate (Step 1) Synthesis of Synthesis Example Compound 35

According to Synthesis Example 30, 1.10 mg of the title compound wasobtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 4-amino-2,3-difluorophenol (7 mg).

Synthesis Example 36 Synthesis of(R)-4-(1-(1-acryloyoypiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-3-methoxyphenyldimethylcarbamate (Step 1) Synthesis of Synthesis Example Compound 36

According to Synthesis Example 30, 10.8 mg of the title compound wasobtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (25 mg) and 4-amino-3-methoxyphenol (17 mg)

Synthesis Example 37 Synthesis of(R)-8-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)quinolin-5-yl dimethylcarbamate (Step 1) Synthesis of8-nitroquinolin-5-yl dimethylcarbamate

To 8-nitroquinolin-5-ol (100 mg), potassium carbonate (220 mg) andacetonitrile (2.6 mL) were added, and N,N-dimethylcarbamoyl chloride(113 mg) was added to the suspension. The mixture was stirred at 60° C.for 2 hours, and then, ethyl acetate and water were added thereto toseparate an organic layer: The organic layer was dried over anhydroussodium sulfate, and the solvent was distilled off under reducedpressure. The residue was purified by silica gel chromatography(hexane/ethyl acetate) to obtain 110 mg of the title compound as ayellow solid.

(Step 2) Synthesis of 8-aminoquinolin-5-yl dimethylcarbamate

According to (Step 2) of Synthesis Example 6, 90 mg of the titlecompound was obtained as a yellow oil by using 8-nitroquinolin-5-yldimethylcarbamate (110 mg) obtained in (Step 1) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 37

According to (Step 3) of Synthesis Example 4, 5.37 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 8-aminoquinolin-5-yl dimethylcarbamate (10 mg) obtainedin (Step 2).

Synthesis Example 38 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-2,5-dimethylphenyldimethylcarbamate (Step 1) Synthesis of Synthesis Example Compound 38

According to Synthesis Example 30, 8.01 mg of the title compound wasobtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 4-amino-2,5-dimethylphenol (10 mg).

Synthesis Example 39 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,5-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of2-(2,5-dimethyl-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 4, 110 mg of the titlecompound was obtained as a yellow oil by using2-(2,5-dimethyl-4-nitrophenyl)acetic acid (100 mg) instead of3-methyl-4-nitrophenylacetic acid.

(Step 2) Synthesis of2-(4-amino-2,5-dimethylphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 90 mg of the titlecompound was obtained as a yellow oil by using2-(2,5-dimethyl-4-nitrophenyl)-N,N-dimethylacetamide (110 mg) obtainedin (Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 39

According to (Step 3) of Synthesis Example 4, 4.50 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-2,5-dimethylphenyl)-N,N-dimethylacetamide(10 mg) obtained in (Step 2).

Synthesis Example 40 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(2-methoxy-4-nitrophenyl)-N,N-dimethylacetamide

According to the synthesis method described in European Journal ofMedicinal Chemistry, 46 (1), 285-296; 2010, 250 mg of the title compoundwas obtained as a yellow oil by using 2-methoxy-4-nitrobenzaldehyde (300mg) instead of 4-nitrobenzaldehyde.

(Step 2) Synthesis of 2-(4-amino-2-methoxyphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 185 mg of the titlecompound was obtained as a yellow oil by using2-(2-methoxy-4-nitrophenyl)-N,N-dimethylacetamide (250 mg) obtained in(Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 40

According to (Step 3) of Synthesis Example 4, 8.22 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-2-methoxyphenyl)-N,N-dimethylacetamide (10mg) obtained in (Step 2).

Synthesis Example 41 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)quinolin-8-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of N,N-dimethyl-2-(8-nitroquinolin-5-yl) acetamide

According to (Step 1) of Synthesis Example 5, 150 mg of the titlecompound was obtained as a yellow solid by using 2-(quinolin-5-yl)aceticacid (220 mg) instead of 1-naphthylacetic acid.

(Step 2) Synthesis of 2-(8-aminoquinolin-5-yl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 110 mg of the titlecompound was obtained as a yellow oil by usingN,N-dimethyl-2-(8-nitroquinolin-5-yl)acetamide (150 mg) obtained in(Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 41

According to (Step 3) of Synthesis Example 4, 12.8 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(8-aminoquinolin-5-yl)-N,N-dimethylacetamide (10 mg)obtained in (Step 2).

Synthesis Example 42 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(2-fluoro-4-nitrophenyl)-N,N-dimethylacetamide

According to the synthesis method described in European Journal ofMedicinal Chemistry, 46 (1), 285-296; 2010, 420 mg of the title compoundwas obtained as a yellow oil by using 2-fluoro-4-nitrobenzaldehyde (550mg) instead of 4-nitrobenzaldehyde.

(Step 2) Synthesis of 2-(4-amino-2-fluorophenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 320 mg of the titlecompound was obtained as a yellow oil by using2-(2-fluoro-4-nitrophenyl)-N,N-dimethylacetamide (420 mg) obtained in(Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 42

According to (Step 3) of Synthesis Example 4, 8.3 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-2-fluorophenyl)-N,N-dimethylacetamide (10mg) obtained in (Step 2).

Synthesis Example 43 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-2,6-difluorophenyldimethylcarbamate (Step 1) Synthesis of 2,6-difluoro-4-nitrophenyldimethylcarbamate

According to (Step 1) of Synthesis Example 29, 260 mg of the titlecompound was obtained as a yellow oil by using2,6-difluoro-4-nitrophenol (200 mg) instead of 2-chloro-4-nitrophenol.

(Step 2) Synthesis of 4-amino-2,6-difluorophenyl dimethylcarbamate

According to (Step 2) of Synthesis Example 6, 220 mg of the titlecompound was obtained as a yellow oil by using2,6-difluoro-4-nitrophenyl dimethylcarbamate (260 mg) obtained in(Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 43

According to (Step 3) of Synthesis Example 4, 7.47 mg of the titlecompound was obtained as a brown solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 4-amino-2,6-difluorophenyl dimethylcarbamate (10 mg)obtained in (Step 2).

Synthesis Example 44 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-3,5-dimethylphenyldimethylcarbamate (Step 1) Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-hydroxy-2,6-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

According to (Step 1) of Synthesis Example 26, 30 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (30 mg) and 4-amino-3,5-dimethylphenol (25 mg).

(Step 2) Synthesis of Synthesis Example Compound 44

According to (Step 2) of Synthesis Example 26, 12 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-hydroxy-2,6-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(10 mg) obtained in (Step 1) instead of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-hydroxy-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.

Synthesis Example 45 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-2,6-dichlorophenyldimethylcarbamate (Step 1) Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3,5-dichloro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

According to (Step 1) of Synthesis Example 26, 20 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (30 mg) and 4-amino-2,6-dichlorophenol (25 mg).

(Step 2) Synthesis of Synthesis Example Compound 45

According to (Step 2) of Synthesis Example 26, 7.5 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3,5-dichloro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(10 mg) obtained in (Step 1) instead of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-hydroxy-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.

Synthesis Example 46 Synthesis of(R)-4-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-2,6-dimethylphenyldimethylcarbamate (Step 1) Synthesis of 2,6-dimethyl-4-nitrophenyldimethylcarbamate

According to (Step 1) of Synthesis Example 29, 260 mg of the titlecompound was obtained as a yellow oil by using2,6-dimethyl-4-nitrophenol (200 mg) instead of 2-chloro-4-nitrophenol.

(Step 2) Synthesis of 4-amino-2,6-dimethylphenyl dimethylcarbamate

According to (Step 2) of Synthesis Example 6, 210 mg of the titlecompound was obtained as a yellow oil by using2,6-dimethyl-4-nitrophenyl dimethylcarbamate (260 mg) obtained in(Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 46

According to (Step 3) of Synthesis Example 4, 13.2 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 4-amino-2,6-dimethylphenyl dimethylcarbamate (10 mg)obtained in (Step 2).

Synthesis Example 47 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-(dimethylamino)-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of2-(3-fluoro-4-nitrophenyl)-N,N-dimethyl-2-acetamide

According to the synthesis method described in European Journal ofMedicinal Chemistry, 46 (1), 285-296; 2010, 300 mg of the title compoundwas obtained as a yellow oil by using 3-fluoro-4-nitrobenzaldehyde (500mg) instead of 4-nitrobenzaldehyde.

(Step 2) Synthesis of 2-(4-amino-3-fluorophenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 240 mg of the titlecompound was obtained as a yellow oil by using2-(3-fluoro-4-nitrophenyl)-N,N-dimethyl-2-acetamide (300 mg) obtained in(Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 47

According to (Step 3) of Synthesis Example 4, 10.2 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-3-fluorophenyl)-N,N-dimethylacetamide (10mg) obtained in (Step 2).

Synthesis Example 48 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-vinylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of N,N-dimethyl-2-(4-nitro-3-vinylphenyl)acetamide

To 2-(3-bromo-4-nitrophenyl)-N,N-dimethylacetamide (330 mg) obtained in(Step 1) of Synthesis Example 17, potassium vinyltrifluoroborate (330mg), a Pd-dppf complex (93 mg), DME (5.7 mL), and a 2 M aqueous sodiumcarbonate solution (2.9 mL) were added. The mixture was stirred at 80°C. for 12 hours, and then, ethyl acetate and water were added thereto toseparate an organic layer. The organic layer was dried over anhydroussodium sulfate, and the solvent was distilled off under reducedpressure. The residue was purified by silica gel chromatography(hexane/ethyl acetate) to obtain 270 mg of the title compound as ayellow solid.

(Step 2) Synthesis of 2-(4-amino-3-vinylphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 80 mg of the titlecompound was obtained as a yellow oil by usingN,N-dimethyl-2-(4-nitro-3-vinylphenyl)acetamide (100 mg) obtained in(Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 48

According to (Step 3) of Synthesis Example 4, 8.16 mg of the titlecompound was obtained as a green solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-3-vinylphenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 2).

Synthesis Example 49 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-ethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(4-amino-3-ethylphenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 4, 80 mg of the titlecompound was obtained as a yellow oil by usingN,N-dimethyl-2-(4-nitro-3-vinylphenyl)acetamide (100 mg) obtained in(Step 1) of Synthesis Example 48 instead of2-(3-methyl-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 2) Synthesis of Synthesis Example Compound 49

According to (Step 3) of Synthesis Example 4, 11.0 mg of the titlecompound was obtained as a yellow solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-3-ethylphenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 1).

Synthesis Example 50 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 2-(3-chloro-2-fluoro-4-nitrophenyl)acetic Acid

To diethyl malonate (621 mg), NMP (5.0 mL), sodium hydride (248 mg), and2-chloro-3,4-difluoro-1-nitrobenzene (500 mg) were added in this orderat room temperature. The solution was heated to 100° C., stirred for 10minutes, and then cooled to room temperature, and ethyl acetate and asaturated aqueous solution of ammonium chloride were added thereto toseparate an organic layer. The organic layer was washed with water andsaturated saline and then dried over anhydrous sodium sulfate, and thesolvent was distilled off under reduced pressure. The residue waspurified by silica gel chromatography (hexane/ethyl acetate) to obtain1.00 g of a yellow oil. Next, methanol (5.0 mL) and a 5 M aqueous sodiumhydroxide solution (2.5 mL) were added to the obtained oil, and themixture was heated to 70° C. and stirred for 1.5 hours. the solution wascooled to room temperature, the solvent was distilled off under reducedpressure, and a 5 M aqueous hydrochloric acid solution was added to theresidue. The precipitated solid was collected by filtration and dried toobtain 366 mg of the title compound as a yellow solid.

(Step 2) Synthesis of2-(3-chloro-2-fluoro-4-nitrophenyl)-N,N-dimethylacetamide

2-(3-Chloro-2-fluoro-4-nitrophenyl)acetic acid (366 mg) obtained in(Step 1) was dissolved by adding DMF (7.85 mL), DIPEA (3.14 mL), and a2.0 M solution of dimethylamine in THF (3.14 mL). To the solution, HATU(895 mg) was then added. The mixture was stirred at room temperature for1 hour, and then, ethyl acetate and water were added thereto to separatean organic layer. The organic layer was washed with water and saturatedsaline and then dried over anhydrous sodium sulfate, and the solvent wasdistilled off under reduced pressure. The residue was purified by silicagel chromatography (hexane/ethyl acetate) to obtain 366 mg of the titlecompound as a yellow solid.

(Step 3) Synthesis of2-(4-amino-3-chloro-2-fluorophenyl)-N,N-dimethylacetamide

To 2-(3-chloro-2-fluoro-4-nitrophenyl)-N,N-dimethylacetamide (150 mg)obtained in (Step 2), THF (1.5 mL) and water (1.5 mL) were added, andthen, ammonium chloride (150 mg) and iron powder (150 mg) were added.The solution was heated to 70° C., stirred for 2 hours, then cooled toroom temperature, and filtered through celite to remove iron. Thesolvent in the filtrate was distilled off under reduced pressure, andthe obtained residue was purified by silica gel chromatography (ethylacetate/methanol) to obtain 120 mg of the title compound as a yellowsolid.

(Step 4) Synthesis of Synthesis Example Compound 50

(R)-1-(1-Acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) obtained in Reference Example 3 and2-(4-amino-3-chloro-2-fluorophenyl)-N,N-dimethylacetamide (30 mg)obtained in (Step 3) were dissolved by adding DMSO (0.2 mL) and DIPEA(10 μL). To the solution, HATU (50 mg) was then added. The mixture wasstirred at room temperature for 1 hour. Then, DMSO (1 mL) was addedthereto, and the mixture was purified by reverse-phase preparative HPLCpurification (water/acetonitrile (0.1% formic acid)) to obtain 6.7 mg ofthe title compound as a white solid.

Synthesis Example 51 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-(furan-2-yl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of2-(3-(furan-2-yl)-4-nitrophenyl)N,N-dimethylacetamide

To 2-(3-bromo-4-nitrophenyl)-N,N-dimethylacetamide (50 mg) obtained in(Step 1) of Synthesis Example 17, 2-furylboronic acid (30 mg), a Pd-dppfcomplex (14 mg), DME (0.9 mL), and a 2 M aqueous sodium carbonatesolution (0.45 mL) were added. The mixture was stirred at 80° C. for 12hours, and then, ethyl acetate and water were added thereto to separatean organic layer. The organic layer was dried over anhydrous sodiumsulfate, and the solvent was distilled off under reduced pressure. Theresidue was purified by silica gel chromatography (hexane/ethyl acetate)to obtain 45 mg of the title compound as a yellow solid.

(Step 2) Synthesis of2-(4-amino-3-(furan-2-yl)phenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 30 mg of the titlecompound was obtained as a yellow oil by using2-(3-(furan-2-yl)-4-nitrophenyl)-N,N-dimethylacetamide (45 mg) obtainedin (Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 51

According to (Step 3) of Synthesis Example 4, 8.09 mg of the titlecompound was obtained as a brown solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-3-(furan-2-yl)phenyl)-N,N-dimethylacetamide(10 mg) obtained in (Step 2).

Synthesis Example 52 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-(furan-3-yl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of2-(3-(furan-3-yl)-4-nitrophenyl)-N,N-dimethylacetamide

According to (Step 1) of Synthesis Example 51, 45 mg of the titlecompound was obtained as a yellow oil by using 3-furylboronic acid (30mg) instead of 2-furylboronic acid.

(Step 2) Synthesis of2-(4-amino-3-(furan-3-yl)phenyl)-N,N-dimethylacetamide

According to (Step 2) of Synthesis Example 6, 30 mg of the titlecompound was obtained as a yellow oil by using2-(3-(furan-3-yl)-4-nitrophenyl)-N,N-dimethylacetamide (45 mg) obtainedin (Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 52

According to (Step 3) of Synthesis Example 4, 4.23 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-3-(furan-3-yl)phenyl)-N,N-dimethylacetamide(10 mg) obtained in (Step 2).

Synthesis Example 53 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(7-(2-(dimethylamino)-2-oxoethyl)benzo[d][1,3]dioxol-4-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of N,N-dimethyl-2-(7-nitrobenzo[d][1,3]dioxol-4-yl)acetamide

To 7-nitrobenzo[d][1,3]dioxole-4-carbaldehyde (180 mg), dichloromethane(4.6 mL), carbon tetrabromide (1.06 g), and triphenylphosphine (1.55 g)were added, and the mixture was stirred at room temperature. Then, thesolvent was removed with an evaporator. The residue was purified bysilica gel chromatography (hexane/ethyl acetate) to obtain a yellow oil.Next, a 50% aqueous dimethylamine solution (5.0 mL) was added to theobtained oil, and the mixture was heated to 50° C. and stirredovernight. After the solution was cooled to room temperature, ethylacetate and a saturated aqueous solution of ammonium chloride were addedthereto to separate an organic layer. The organic layer was washed withwater and saturated saline and then dried over anhydrous sodium sulfate,and the solvent was distilled off under reduced pressure. The residuewas purified by silica gel chromatography (hexane/ethyl acetate) toobtain 120 mg of the title compound as a yellow oil.

(Step 2) Synthesis of2-(7-aminobenzo[d][1,3]dioxol-4-yl)-N,N-dimethylacetamide

According to (Step 3) of Synthesis Example 1, 100 mg of the titlecompound was obtained as a yellow oil by usingN,N-dimethyl-2-(7-nitrobenzo[d][1,3]dioxol-4-yl)acetamide (120 mg)obtained in (Step 1) instead of2-(2-bromo-5-methyl-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 53

According to (Step 4) of Synthesis Example 1,(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(7-aminobenzo[d][,3]dioxol-4-yl)-N,N-dimethylacetamide (20 mg) obtained in (Step 2) weredissolved by adding DMSO (0.2 mL) and DIPEA (20 μL). To the solution,HATU (50 mg) was then added to obtain 6.38 mg of the title compound as awhite solid.

Synthesis Example 54 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of Synthesis Example Compound 54

(R)-1-(1-Acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) synthesized in Reference Example 5 was dissolved by adding2-(4-amino-3-chlorophenyl)-N,N-dimethylacetamide (10 mg) obtained in(Step 2) of Synthesis Example 6, DMF (0.2 mL), and DIPEA (20 μL). To thesolution, HATU (18 mg) was then added. The mixture was stirred at roomtemperature for 1 hour. Then, DMSO (1 mL) was added thereto, and themixture was purified by reverse-phase preparative HPLC purification(water/acetonitrile (0.1% formic acid)) to obtain 2.8 mg of the titlecompound as a white solid.

Synthesis Example 55 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of Synthesis Example Compound 55

According to (Step 1) of Synthesis Example 54, 3.33 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (5 mg) and2-(4-amino-2-chloro-3-methoxyphenyl)-N,N-dimethylacetamide (5 mg)synthesized in (Step 3) of Synthesis Example 11.

Synthesis Example 56 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of Synthesis Example Compound 56

According to (Step 1) of Synthesis Example 54, 2.19 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (5 mg) and2-(4-amino-2-fluoro-3-methylphenyl)-N,N-dimethylacetamide (5 mg)synthesized in (Step 3) of Synthesis Example 12.

Synthesis Example 57 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of Synthesis Example Compound 57

According to (Step 1) of Synthesis Example 54, 4.25 mg of the titlecompound was obtained as a brown solid by using(R)-1-(1-acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (5 mg) and2-(4-amino-2-fluoro-3-methoxyphenyl)-N,N-dimethylacetamide (5 mg)synthesized in (Step 3) of Synthesis Example 14.

Synthesis Example 58 Synthesis of(R)-4-(1-(1-acryloylpyrrolidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)naphthalen-1-yl dimethylcarbamate (Step 1) Synthesis of4-nitronaphthalen-1-yl dimethylcarbamate

According to (Step 1) of Synthesis Example 29, the title compound wasobtained as a yellow solid by using 4-nitronaphthol instead of2-chloro-4-nitrophenol.

(Step 2) Synthesis of 4-aminonaphthalen-1-yl dimethylcarbamate

According to (Step 2) of Synthesis Example 6, the title compound wasobtained as a yellow oil by using 4-nitronaphthalen-1-yldimethylcarbamate obtained in (Step 1) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 58

According to (Step 1) of Synthesis Example 54, 3.59 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (7 mg) and 4-aminonaphthalen-1-yl dimethylcarbamate (7 mg)synthesized in (Step 2) of Synthesis Example 58.

Synthesis Example 59 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of Synthesis Example Compound 59

According to (Step 1) of Synthesis Example 54, 1.23 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (5 mg) and 2-(4-amino-2,3-dimethylphenyl)-N,N-dimethylacetamide (5mg) synthesized in (Step 3) of Synthesis Example 13.

Synthesis Example 60 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-vinylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of Synthesis Example Compound 60

According to (Step 1) of Synthesis Example 54, 2.82 mg of the titlecompound was obtained as a brown solid by using(R)-1-(1-acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (5 mg) and 2-(4-amino-3-vinylphenyl)-N,N-dimethylacetamide (5 mg)synthesized in (Step 2) of Synthesis Example 48.

Synthesis Example 61 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (Step 1)Synthesis of Synthesis Example Compound 61

According to (Step 1) of Synthesis Example 54, 5.3 mg of the titlecompound was obtained as a yellow solid by using(R)-1-(1-acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (5 mg) and 2-(4-aminonaphthalen-1-yl)-N,N-dimethylacetamide (5 mg)synthesized in (Step 2) of Synthesis Example 5.

Synthesis Example 62 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(2-bromo-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of Synthesis Example Compound 62

According to (Step 1) of Synthesis Example 54, 0.90 mg of the titlecompound was obtained as a brown solid by using(R)-1-(1-acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (5 mg) and 2-(4-amino-3-bromophenyl)-N,N-dimethylacetamide (5 mg)synthesized in (Step 2) of Synthesis Example 17.

Synthesis Example 63 Synthesis of(R)-4-(1-(1-acryloylpyrrolidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-3-methylphenyldimethylcarbamate (Step 1) Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(4-hydroxy-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

(R)-1-(1-Acryloylpyrrolidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) synthesized in Reference Example 5 was dissolved by addingDMF (0.2 mL), 4-amino-3-methylphenol (5.8 mg), and DIPEA (20 μL). To thesolution, HATU (17 mg) was then added. The mixture was stirred at roomtemperature for 1 hour, and then, ethyl acetate and water were addedthereto to separate an organic layer. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off underreduced pressure. The residue was purified by silica gel chromatography(chloroform/methanol) to obtain 13 mg of the title compound as acolorless oil.

(Step 2) Synthesis of Synthesis Example Compound 63

To(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(4-hydroxy-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(13 mg) obtained in (step 1), potassium carbonate (10 mg) andacetonitrile (250 μL) were added to prepare a suspension. Then,N,N-dimethylcarbamoyl chloride (8 mg) was added thereto. The mixture wasstirred at 60° C. for 3 hours, and then, chloroform and water were addedthereto to separate an organic layer. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off underreduced pressure. The residue was purified by reverse-phase preparativeHPLC purification (water/acetonitrile (0.1% formic acid)) to obtain 6.10mg of the title compound as a white solid.

Synthesis Example 64 Synthesis of(R)-4-(1-(1-acryloylpyrrolidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-2,3-dimethylphenyldimethylcarbamate (Step 1) Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(4-hydroxy-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

According to (Step 1) of Synthesis Example 63, 10 mg of the titlecompound was obtained as a white solid by using4-amino-2,3-dimethylphenol instead of 4-amino-3-methylphenol.

(Step 2) Synthesis of Synthesis Example Compound 64

According to (Step 2) of Synthesis Example 63, 6.70 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(4-hydroxy-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(10 mg) obtained in (Step 1) instead of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(4-hydroxy-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.

Synthesis Example 65 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of Synthesis Example Compound 65

(R)-1-(1-Acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (5.0 mg) obtained in Reference Example 5 and2-(4-amino-3-chloro-2-fluorophenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 3) of Synthesis Example 50 were dissolved by addingDMSO (0.1 mL) and DIPEA (5 μL). To the solution, HATU (25 mg) was thenadded. The mixture was stirred at room temperature for 1 hour. Then,DMSO (1 mL) was added thereto, and the mixture was purified byreverse-phase preparative HPLC purification (water/acetonitrile (0.1%formic acid)) to obtain 3.41 mg of the title compound as a white solid.

Synthesis Example 66 Synthesis of(R,E)-4-(4-amino-1-(1-(4-dimethylamino) but-2-enoyl)piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)naphthalen-1-yldimethylcarbamate (Step 1) Synthesis of (R)-tert-butyl3-(4-amino-3((4-((dimethylcarbamoyl)oxy)naphthalen-1-yl)carbamoyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate

(R)-4-Amino-1-(1-(tert-butyloxycarbonyl)piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (20 mg) synthesized in Reference Example 2 was dissolved by adding4-aminonaphthalene-1-yldimethylcarbamate (20 mg) synthesized in (Step 2)of Synthesis Example 58, DMF (0.2 mL), and DIPEA (35 μL). To thesolution, HATU (32 mg) was then added, and the mixture was stirred atroom temperature for 1 hour. Ethyl acetate and water were added theretoto separate an organic layer. The organic layer was dried over anhydroussodium sulfate, and the solvent was distilled off under reducedpressure. The residue was purified by silica gel chromatography(chloroform/methanol) to obtain 20 mg of the title compound as acolorless oil.

(Step 2) Synthesis of Synthesis Example Compound 66

To (R)-tert-butyl 3-(4-amino-3 ((4-((dimethylcarbamoyl)oxy)naphthalen-1-yl) carbamoyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (20 mg) obtained in (Step 1), a 4 M solution ofhydrochloric acid in 1,4-dioxane (0.3 mL) was added, and the mixture wasstirred at room temperature for 1 hour. The organic solvent wasdistilled off under reduced pressure, and then, the residue wasdissolved by adding trans-4-dimethylaminocrotonic acid (5 mg), DMF (0.2mL), and DIPEA (20 μL). To the solution, HATU (15 mg) was then added,and the mixture was stirred at room temperature for 1 hour. DMSO (1.0mL) was added to the solution, and the mixture was purified byreverse-phase preparative HPLC purification (water/acetonitrile (0.1%formic acid)) to obtain 12.7 mg of the title compound as a white solid.

Synthesis Example 67 Synthesis of(R)-1-(1-acryloylpyrrolidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

(R)-1-(1-Acryloylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (5.0 mg) obtained in Reference Example 5 and2-(4-amino-2-chloro-5-methylphenyl)-N,N-dimethylacetamide (10 mg)obtained in (Step 4) of Synthesis Example 19 were dissolved by addingDMSO (0.1 mL) and DIPEA (5 μL). To the solution, HATU (25 mg) was thenadded. The mixture was stirred at room temperature for 1 hour. Then,DMSO (1 mL) was added thereto, and the mixture was purified byreverse-phase preparative HPLC purification (water/acetonitrile (0.1%formic acid)) to obtain 5.08 mg of the title compound as a white solid.

Synthesis Example 68 Synthesis of(R)-4-(1-(1-acryloylpyrimidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-3-methylphenylmethyl(phenyl) carbamate (Step 1) Synthesis of 3-methyl-4-nitrophenylmethyl(phenyl) carbamate

According to (Step 1) of Synthesis Example 29, 80 mg of the titlecompound was obtained as a yellow solid by using 3-methyl-4-nitrophenol(50 mg) instead of 2-chloro-4-nitrophenol and usingN-methyl-N-phenylcarbamoyl chloride instead of N,N-dimethylcarbamoylchloride.

(Step 2) Synthesis of 4-amino-3-methylphenyl methyl(phenyl)carbamate

According to (Step 2) of Synthesis Example 6, 50 mg of the titlecompound was obtained as a yellow oil by using 3-methyl-4-nitrophenylmethyl(phenyl)carbamate (80 mg) obtained in (Step 1) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 68

According to (Step 3) of Synthesis Example 4, 3.58 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (7.5 mg) and 4-amino-3-methylphenyl methyl(phenyl)carbamate (7.5mg) obtained in (Step 2).

Synthesis Example 69 Synthesis of(R)-4-(1-(1-acryloylpyrimidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-3-methylphenylpyrrolidine-1-carboxylate (Step 1) Synthesis of 3-methyl-4-nitrophenylpyrrolidine-1-carboxylate

According to (Step 1) of Synthesis Example 29, 80 mg of the titlecompound was obtained as a yellow solid by using 3-methyl-4-nitrophenol(50 mg) instead of 2-chloro-4-nitrophenol and using1-pyrrolidinecarboxylic acid chloride instead of N,N-dimethylcarbamoylchloride.

(Step 2) Synthesis of 4-amino-3-methylphenyl pyrrolidine-1-carboxylate

According to (Step 2) of Synthesis Example 6, 50 mg of the titlecompound was obtained as a yellow oil by using 3-methyl-4-nitrophenylpyrrolidine-1-carboxylate (80 mg) obtained in (Step 1) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 69

According to (Step 3) of Synthesis Example 4, 3.66 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (7.5 mg) and 4-amino-3-methylphenyl pyrrolidine-1-carboxylate (7.5mg) obtained in (Step 2).

Synthesis Example 70 Synthesis of(R)-4-(1-(1-acryloylpyrimidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamido)-3-methylphenylpiperidine-1-carboxylate (Step 1) Synthesis of 3-methyl-4-nitrophenylpiperidine-1-carboxylate

According to (Step 1) of Synthesis Example 29, 70 mg of the titlecompound was obtained as a yellow solid by using 3-methyl-4-nitrophenol(50 mg) instead of 2-chloro-4-nitrophenol and using1-piperidinecarboxylic acid chloride instead of N,N-dimethylcarbamoylchloride.

(Step 2) Synthesis of 4-amino-3-methylphenyl pyrrolidine-1-carboxylate

According to (Step 2) of Synthesis Example 6, 50 mg of the titlecompound was obtained as a yellow oil by using 3-methyl-4-nitrophenylpiperidine-1-carboxylate (70 mg) obtained in (Step 1) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 70

According to (Step 3) of Synthesis Example 4, 4.05 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (7.5 mg) and 4-amino-3-methylphenyl pyrrolidine-1-carboxylate (7.5mg) obtained in (Step 2).

Synthesis Example 71 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-methyl-4-(2-oxo-2-(pyrrolidin-1-yl)ethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of2-(3-methyl-4-nitrophenyl)-1-(pyrrolidin-1-yl)ethanone

According to (Step 1) of Synthesis Example 4, 55 mg of the titlecompound was obtained as a yellow oil by using pyrrolidine instead of a2.0 M solution of dimethylamine in THF.

(Step 2) Synthesis of2-(4-amino-3-methylphenyl)-1-(pyrrolidin-1-yl)ethanone

According to (Step 2) of Synthesis Example 6, 50 mg of the titlecompound was obtained as a yellow oil by using2-(3-methyl-4-nitrophenyl)-1-(pyrrolidin-1-yl)ethanone (55 mg) obtainedin (Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 71

According to (Step 3) of Synthesis Example 4, 3.49 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-3-methylphenyl)-1-(pyrrolidin-1-yl)ethanone(10 mg) obtained in (Step 2).

Synthesis Example 72 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-methoxy(methyl)amino-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis ofN-methoxy-N-methyl-2-(3-methyl-4-nitrophenyl)acetamide

According to (Step 1) of Synthesis Example 4, 55 mg of the titlecompound was obtained as a yellow oil by using N-methoxy-N-methylamineinstead of a 2.0 M solution of dimethylamine in THF.

(Step 2) Synthesis of2-(4-amino-3-methylphenyl)-N-methoxy-N-methylacetamide

According to (Step 2) of Synthesis Example 6, 50 mg of the titlecompound was obtained as a yellow oil by usingN-methoxy-N-methyl-2-(3-methyl-4-nitrophenyl)acetamide (55 mg) obtainedin (Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 72

According to (Step 3) of Synthesis Example 4, 4.43 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 2-(4-amino-3-methylphenyl)-N-methoxy-N-methylacetamide(10 mg) obtained in (Step 2).

Synthesis Example 73 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of1-(3-hydroxyazetidin-1-yl)-2-(3-methyl-4-nitrophenyl)ethanone

According to (Step 1) of Synthesis Example 4, 55 mg of the titlecompound was obtained as a yellow oil by using 3-hydroxyazetidineinstead of a 2.0 M solution of dimethylamine in THF.

(Step 2) Synthesis of2-(4-amino-3-methylphenyl)-1-(3-hydroxyazetidin-1-yl)ethanone

According to (Step 2) of Synthesis Example 6, 50 mg of the titlecompound was obtained as a yellow oil by using1-(3-hydroxyazetidin-1-yl)-2-(3-methyl-4-nitrophenyl)ethanone (55 mg)obtained in (Step 1) instead of2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 73

According to (Step 3) of Synthesis Example 4, 8.14 mg of the titlecompound was obtained as a brown solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and2-(4-amino-3-methylphenyl)-1-(3-hydroxyazetidin-1-yl)ethanone (10 mg)obtained in (Step 2).

Synthesis Example 74 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(3,3-dimethylureido)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of 1,1-dimethyl-3-(3-methyl-4-nitrophenyl)urea

3-Methyl-4-nitroaniline (100 mg) was dissolved by adding DMF (3.3 mL)and DIPEA (170 μL). To the solution, dichloromethylene methyliminiumchloride (125 mg) was then added. The mixture was stirred at 50° C. for3 hours, and then, ethyl acetate and water were added thereto toseparate an organic layer. The organic layer was dried over anhydroussodium sulfate, and the solvent was distilled off under reducedpressure. The residue was purified by silica gel chromatography(chloroform/methanol) to obtain 140 mg of the title compound as acolorless oil.

(Step 2) Synthesis of 3-(4-amino-3-methylphenyl)-1,1-dimethylurea

According to (Step 2) of Synthesis Example 6, 120 mg of the titlecompound was obtained as a yellow oil by using1,1-dimethyl-3-(3-methyl-4-nitrophenyl)urea (140 mg) obtained in(Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 74

According to (Step 3) of Synthesis Example 4, 6.50 mg of the titlecompound was obtained as a brown solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and 3-(4-amino-3-methylphenyl)-1,1-dimethylurea (10 mg)obtained in (Step 2).

Synthesis Example 75 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-methyl-4-(pyrrolidine-1-carboxamido)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis ofN-(3-methyl-4-nitrophenyl)pyrrolidine-1-carboxamide

3-Methyl-4-nitroaniline (30 mg) was dissolved by adding DMF (2.0 mL) andsodium hydride (60%) (12 mg). To the solution, 1-pyrrolidinecarboxylicacid chloride (25 μL) was then added. The mixture was stirred at roomtemperature for 30 minutes, and then, ethyl acetate and water were addedthereto to separate an organic layer. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off underreduced pressure. The residue was purified by silica gel chromatography(chloroform/methanol) to obtain 30 mg of the title compound as acolorless oil.

(Step 2) Synthesis ofN-(4-amino-3-methylphenyl)pyrrolidine-1-carboxamide

According to (Step 2) of Synthesis Example 6, 25 mg of the titlecompound was obtained as a yellow oil by usingN-(3-methyl-4-nitrophenyl)pyrrolidine-1-carboxamide (30 mg) obtained in(Step 1) instead of 2-(3-chloro-4-nitrophenyl)-N,N-dimethylacetamide.

(Step 3) Synthesis of Synthesis Example Compound 75

According to (Step 3) of Synthesis Example 4, 6.53 mg of the titlecompound was obtained as a white solid by using(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (10 mg) and N-(4-amino-3-methylphenyl)pyrrolidine-1-carboxamide (10mg) obtained in (Step 2).

Synthesis Example 76 Synthesis of1-((3R)-1-acryloyl-5-fluoropiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of (2S,4R)-methyl l-benzyl-4-(benzyloxy)pyrrolidine-2-carboxylate

To diethyl ether (400 mL), (2S,4R)-methyl4-(benzyloxy)pyrrolidine-2-carboxylate hydrochloride (4.57 g) was added,and then triethylamine (2.98 g) was added under ice cooling. The mixturewas stirred under ice cooling for 30 minutes. Then, benzyl bromide (5.04g) and triethylamine (2.98 g) were added thereto, and the mixture wasstirred overnight at room temperature. The obtained suspension wasfiltered, and then, the filtrate was washed with water and saturatedsaline, then dried over anhydrous sodium sulfate, and filtered. Afterconcentration, the obtained residue was purified by silica gelchromatography (hexane/ethyl acetate) to obtain 4.57 g of the titlecompound as a colorless oil.

(Step 2) Synthesis of ((2S,4R)-methyll-benzyl-4-(benzyloxy)pyrrolidin-2-yl)methanol

Lithium aluminum hydride (1.99 g) was suspended in THF (100 mL). To thesuspension, a solution of (2S,4R)-methyll-benzyl-4-(benzyloxy)pyrrolidine-2-carboxylate (11.4 g) in THF (65 mL)was added under ice cooling. The mixture was kept been stirred under icecooling for 1 hour, and then, sodium sulfate decahydrate (20 g) wasadded thereto. The mixture was stirred at room temperature for 3 hours.Then, anhydrous sodium sulfate decahydrate (20 g) was added thereto, andthe mixture was stirred overnight at room temperature. Anhydrous sodiumsulfate (20 g) was further added thereto, and the mixture was stirredfor 30 minutes, then filtered through celite, and concentrated to obtaina residue in a yellow oil form. This residue was purified by silica gelchromatography (hexane/ethyl acetate) to obtain 9.16 g of the titlecompound as a colorless oil.

(Step 3) Synthesis of (3R)-1-benzyl-3-(benzyloxy)-5-fluoropiperidine

To a solution of ((2S,4R)-methyll-benzyl-4-(benzyloxy)pyrrolidin-2-yl)methanol (9.16 g) in THR,bis(2-methoxyethyl)aminosulfur trifluoride (6.95 g) was added under icecooling, and then, the mixture was kept been stirred for 1 hour. Then,the mixture was stirred at room temperature for 1.5 hours. A saturatedaqueous solution of sodium bicarbonate was added thereto, followed byextraction with ethyl acetate. Then, the extract was washed withsaturated saline, dried using anhydrous sodium sulfate, filtered, andconcentrated. The obtained residue was purified by silica gelchromatography (hexane/ethyl acetate) to obtain 6.08 g of the titlecompound as a colorless oil.

(Step 4) Synthesis of(SR)-tert-butyl-3-fluoro-5-hydroxypiperidine-1-carboxylate

(3R)-1-Benzyl-3-(benzyloxy)-5-fluoropiperidine (6.08 g) anddi-tert-butyl dicarbonate (4.88 g) were dissolved in ethanol (120 mL).To the solution, palladium hydroxide-active carbon (900 mg) was thenadded, and the mixture was stirred for 2 days in a hydrogen atmosphere.The suspension was filtered through celite and then concentrated, andthe residue was suspended and washed using hexane to obtain 3.98 g ofthe title compound as a colorless solid.

(Step 5) Synthesis of(5S)-tert-butyl-3-fluoro-5-((4-nitrobenzoyl)oxy)piperidine-1-carboxylate

(5R)-tert-Butyl-3-fluoro-5-hydroxypiperidine-1-carboxylate (1.0 g),4-nitrobenzoic acid (1.14 g), and triphenylphosphine (1.79 g) weredissolved in toluene (100 mL). To the solution, bis(2-methoxyethyl)azodicarboxylate (1.60 g) was then added under ice cooling. The solutionwas stirred at room temperature for 3 days, then washed with a saturatedaqueous solution of sodium bicarbonate and saturated saline, then driedover anhydrous sodium sulfate, filtered, and then concentrated. Theobtained residue was purified by silica gel chromatography (hexane/ethylacetate) to obtain 1.26 g of the title compound as a colorless solid.

(Step 6) Synthesis of(5S)-tert-butyl-3-fluoro-5-hydroxypiperidine-1-carboxylate

(5S)-tert-Butyl-3-fluoro-5-((4-nitrobenzoyl)oxy)piperidine-1-carboxylate(1.26 g) was dissolved in a mixed solution of THF (7 mL) and methanol (7mL). To the solution, a 5 N aqueous sodium hydroxide solution (1.4 mL)was added.

The mixture was stirred at room temperature for 1.5 hours, and then, theorganic solvent was removed under reduced pressure. The residue wasdiluted with diethyl ether, then washed with water, a saturated aqueoussolution of sodium bicarbonate, and saturated saline, dried overanhydrous sodium sulfate, filtered, and concentrated to obtain the titlecompound (0.69 g) as a colorless solid.

(Step 7) Synthesis of(3R)-tert-butyl-3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-5-fluoropiperidine-1-carboxylate

The title compound (0.33 g) was obtained as a light brown compound byusing (5S)-tert-butyl-3-fluoro-5-hydroxypiperidine-1-carboxylate (0.69g) instead of (S)—N-Boc-3-piperidinol in (Step 1) and (Step 2) ofReference Example 1.

(Step 8) Synthesis of4-amino-1-((3R)-1-(tert-butoxycarbonyl)-5-fluoropiperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicAcid

(3R)-tert-Butyl-3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-5-fluoropiperidine-1-carboxylate(0.300 g), triethylamine (0.32 g), and a1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride-dichloromethane complex (0.052 g) were added to methanol (5mL), and the mixture was stirred at 100° C. for 1 hour at 4.5 atm incarbon monoxide, and then concentrated. The obtained residue waspurified by silica gel chromatography (hexane/ethyl acetate). Theobtained solid was dissolved in methanol (5 mL). To the solution, a 5 Naqueous sodium hydroxide solution (0.64 mL) was added, and then, themixture was stirred at room temperature for 4 hours. Then, a 5 N aqueoushydrochloric acid solution (0.65 mL) was added thereto, followed byextraction with chloroform. Then, the extract was dried over anhydroussodium sulfate, filtered, and concentrated to obtain the title compound(0.21 g) as a colorless solid.

(Step 9) Synthesis of Synthesis Example Compound 76

4-Amino-1-(3R)-1-(tert-butoxycarbonyl)-5-fluoropiperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (35 mg), 2-(4-amino-3-chlorophenyl)-N,N-dimethylacetamide (23 mg)obtained in (Step 2) of Synthesis Example 6, and diisopropylethylamine(23 mg) were dissolved in DMF (1 mL). To the solution, HATU (52 mg) wasadded, and the mixture was stirred for 2 days. After concentration, theresidue was purified by silica gel chromatography and then purified byreverse-phase preparative HPLC purification (water/acetonitrile (0.1%formic acid)). The obtained colorless oil was dissolved in methanol (2mL). To the solution, a solution of 4 N hydrochloric acid in dioxane (2mL) was added, and the mixture was stirred at room temperature for 2hours. After concentration, the obtained yellow solid anddiisopropylethylamine (0.039 g) were dissolved in chloroform (5 mL). Tothe solution, a chloroform solution containing acryloyl chloride (5 mg)was added under ice cooling, and the mixture was stirred for 10 minutes.The solution was purified by silica gel chromatography, concentrated,and then suspended in hexane/ethyl acetate, and the suspension wasfiltered and dried under reduced pressure to obtain the title compound(25 mg) as a colorless solid.

Synthesis Example 77 Synthesis of1-((3R)-1-acryloyl-5-fluoropiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

According to (Step 9) of Synthesis Example 76, the title compound (21mg) was obtained as a colorless solid by using2-(4-amino-2,3-dimethylphenyl)-N,N-dimethylacetamide (22 mg) synthesizedin (Step 3) of Synthesis Example 13 instead of2-(4-amino-3-chlorophenyl)-N,N-dimethylacetamide.

Synthesis Example 78 Synthesis of1-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-N-(2-chloro-4-(2-dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Step 1) Synthesis of (2S,4R)-tert-butyl4-(3-iodo-4-((triphenylphosphoranylidene)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpyrrolidine-1-carboxylate

tert-Butyl (2S,4S)-4-hydroxy-2-methyl-pyrrolidine-1-carboxylate (0.30g), triphenylphosphine, and 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-aminewere dissolved in THF (7 mL). To the solution, diisopropylazodicarboxylate (0.443 mL) was added under ice cooling. The solutionwas stirred at room temperature for 2 hours, and then, THF (10 mL) wasfurther added thereto. After overnight stirring, triphenylphosphine(0.117 g) and diisopropyl azodicarboxylate (0.089 mL) were further addedthereto, and the mixture was further stirred for 1 hour. Afterconcentration of the solution, the residue was suspended in ethylacetate, and the resulting insoluble matter was removed. Then, thefiltrate was concentrated, and the residue was purified by silica gelchromatography (hexane/ethyl acetate) to obtain the title compound(0.387 g) as a colorless amorphous.

(Step 2) Synthesis of (2S,4R)-tert-butyl4-(3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpyrrolidine-1-carboxylate

(2S,4R)-tert-Butyl4-(3-iodo-4-((triphenylphosphoranylidene)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpyrrolidine-1-carboxylate(0.37 g) synthesized in (Step 1) was dissolved in trifluoroacetic acid(5 mL), and the solution was stirred overnight. After concentration,chloroform and water were added to the residue, and then, the mixturewas rendered basic using 5 N sodium hydroxide, followed by extractionwith chloroform/methanol. The obtained organic layer was dried overanhydrous sodium sulfate, filtered, and concentrated. The obtainedcolorless solid was dissolved in chloroform (5 mL). To the solution,di-tert-butyl dicarbonate (0.126 g) was added, and the mixture wasstirred at room temperature for 2 hours. After concentration, theresidue was purified by silica gel chromatography (chloroform/methanol)to obtain the title compound (0.23 g) as a colorless solid.

(Step 3) Synthesis of4-amino-1-((3R,5S)-1-(tert-butoxycarbonyl)-5-methylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicAcid

According to (Step 8) of Synthesis Example 76, the title compound (0.138g) was obtained as a brown solid by using (2S,4R)-tert-butyl4-(3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpyrrolidine-1-carboxylate(0.23 g) obtained in (Step 2) instead of(3R)-tert-butyl-3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-5-fluoropiperidine-1-carboxylate.

(Step 4) Synthesis of Synthesis Example Compound 78

According to (Step 9) of Synthesis Example 76, the title compound (23mg) was obtained as a colorless solid by using4-amino-1-((3R,5S)-1-(tert-butoxycarbonyl)-5-methylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (35 mg) and 2-(4-amino-3-chlorophenyl)-N,N-dimethylacetamide (24mg) obtained in (Step 2) of Synthesis Example 6 instead of4-amino-1-((3R)-1-(tert-butoxycarbonyl)-5-fluoropiperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid.

Synthesis Example 79 Synthesis of1-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-N-(4-(2-dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide

According to (Step 9) of Synthesis Example 76, the title compound (36mg) was obtained as a colorless solid by using4-amino-1-((3R,5S)-1-(tert-butoxycarbonyl)-5-methylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid (35 mg) and 2-(4-amino-2,3-dimethylphenyl)-N,N-dimethylacetamide(24 mg) synthesized in (Step 3) of Synthesis Example 13 instead of4-amino-1-((3R)-1-(tert-butoxycarbonyl)-5-fluoropiperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylicacid.

Hereinafter, a list of Synthesis Example compounds 1 to 79 is shownbelow.

TABLE 1

W

NMR mass 1

CH2

1H-NMR (DMSO-D6) δ: 1.53-1.67 (m, 1.0H), 1.93-2.03 (m, 1.0H), 2.15- 2.52(m, 5.0H), 2.85 (s, 3.0H), 3.03 (s, 3.0H), 3.14-3.89 (m, 2.0H), 3.78 (s,2.0H), 4.00-4.87 (m, 3.0H), 5.58- 5.71 (m, 1.0H), 6.00-6.17 (m, 1.0H),6.68-6.92 (m, 1.0H), 7.20 (s, 1.0H), 7.68-7.76 (m, 1.0H), 8.15 (s,1.0H), 8.27 (s, 1.0H), 8.48 (s, 1.0H), 10.02- 10.22 (m, 1.0H). 569.2 2

CH2

1H NMR (400 MHz, CDCl3) Shift 1.63-1.85 (m, 1H), 1.95-2.10 (m, 1H),2.16-2.41 (m, 5H), 3.01 (s, 3H), 3.05 (s, 3H), 3.18-3.78 (m, 3H), 3.95-4.30 (m, 1H), 4.41-5.02 (m, 2H), 5.61-5.78 (m, 1H), 6.29 (dd, J = 15.85,7.07 Hz, 1H), 6.40-6.72 (m, 2H), 7.23 (s, 1H), 8.21-8.43 (m, 2H), 8.80(br. d, J = 8.00 Hz, 1H), 9.43 (s, 1H) 525.3 3

CH2

1H NMR (400 MHz, CDCl3) Shift 1.15-1.48 (m, 1H), 1.64-1.87 (m, 1H),1.98-2.18 (m, 1H), 2.26-2.34 (m, 2H), 2.37 (s, 3H), 3.02 (s, 3H), 3.08(s, 3H), 3.22-3.56 (m, 1H), 3.73 (s, 2H), 3.96-4.63 (m, 1H), 4.78-5.04(m, 1H), 5.60-5.84 (m, 1H), 6.23-6.73 (m, 3H), 7.00-7.18 (m, 1H),8.10-8.44 (m, 2H), 8.82 (br. d, J = 11.00 Hz, 1H), 9.63 (s, 1H) 525.3 4

CH2

1H NMR (400 MHz, DMSO) Shift 1.58 (br. s., 1H), 1.95 (br. s., 1H),2.12-2.25 (m, 4H), 2.29 (br. s., 1H), 2.81 (d, J = 3.42 Hz, 3H), 2.99(d, J = 4.10 Hz, 3H), 3.63 (br. s., 2H), 4.09 (br. s., 1H), 4.50 (br.s., 1H), 4.70 (br. s., 1H), 5.68 (br. s., 1H), 6.00-6.12 (m, 1H), 6.82(br. s., 1H), 7.03-7.14 (m, 2H), 7.37 (br. s., 1H), 8.06 (br. s., 1H),8.24 (br. s., 1H), 8.53 (br. s., 1H), 10.01 (br. s., 1H) 491.3 5

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.59 (br. s., 1H), 1.96 (br. s., 1H),2.21 (br. s., 1H), 2.38 (br. s., 1H), 2.50 (s, 1H), 2.85 (s, 3H), 3.02(s., 3H), 3.83 (br. s., 1H), 4.06 (br. s., 2H), 4.15 (s, 3H), 4.26 (br.s., 1H), 4.77 (br. s., 1H), 5.58-5.70 (m, 1H), 6.09 (d, J = 16.40 Hz,1H), 6.61-6.94 (m, 1H), 7.35 (d, J = 7.52 Hz, 1H), 7.54 (d, J = 8.88 Hz,3H), 7.83-7.99 (m, 2H), 8.04 (br. s., 1H), 8.25 (s, 1H), 8.50 (br. s.,1H), 10.42- 10.80 (m, 1H) 527.3 6

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.53-1.70 (m, 1H), 1.92-2.03 (m, 1H),2.14-2.37 (m, 2H), 2.84 (s, 3H), 3.03 (s, 3H), 3.18-3.40 (m, 1H),3.68-3.80 (m, 1H), 3.74 (s, 2H), 4.02-4.57 (m, 2H), 4.69-4.81 (m, 1H),5.56-5.71 (m, 1H), 6.03-6.14 (m, 1H), 6.66-6.93 (m, 1H), 7.22-7.27 (m,1H), 7.38-7.46 (m, 1H), 7.74-7.82 (m, 1H), 8.19 (br s, 1H), 8.28 (s,1H), 8.42 (br s, 1H), 10.13 (br s, 1H) 511.2 7

CH2

1H NMR (400 MHz, DMSO) Shift 1.60 (br. s., 1H), 1.95 (br. s., 1H), 2.18(br. s., 1H), 2.27 (d, J = 14.35 Hz, 1H), 2.81 (s, 3H), 2.99 (s, 3H),3.66 (s, 2H), 3.79 (br. s., 1H), 3.87 (s, 3H), 3.99 (d, J = 7.52 Hz,1H), 4.13-4.48 (m, 1H), 4.73 (br. s,1H), 5.48-5.69 (m, 1H), 5.98-6.10(m, 1H), 6.57-6.87 (m, 2H), 6.97 (s, 1H), 8.01 (d, J = 8.20 Hz, 1H),8.15 (br. s., 1H), 8.26 (s, 1H), 8.48 (br. s., 1H), 9.61 (br. s., 1H)507.3 8

CH2

1H NMR (400 MHz, DMSO-d6) Shift 2.15 (br. s., 1H), 2.37-2.55 (m, 1H),2.73 (br. s., 1H), 3.40 (s, 3H), 3.62 (s, 3H), 4.32 (s, 3H), 4.64 (br.s., 2H), 4.70-5.16 (m, 2H), 5.18-5.44 (m, 1H), 6.27 (br. s., 1H), 6.66(br. s., 1H), 7.41 (br. s., 1H), 7.84 (d, J = 8.20 Hz, 1H), 8.29 (d, J =8.88 Hz, 1H), 8.53 (br. s., 1H), 8.67-8.77 (m, 2H), 8.83 (s, 1H), 8.97(br. s., 1H), 10.79-11.13 (m, 1H) 510.3

TABLE 2  9

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.25 (d, J = 6.15 Hz, 1H), 1.55-1.74 (m,1H), 1.99 (s, 1H), 2.22 (m, 1H), 2.28 (br. s., 1H), 2.83-2.90 (m, 3H),3.09 (s, 3H), 3.39-3.59 (m, 1H), 3.79 (s, 2H), 3.92 (s, 3H), 3.96-4.10(m, 1H), 4.15-4.53 (m, 1H), 4.71-4.89 (m, 1H), 5.51-5.76 (m, 1H), 6.08(br. s., 1H), 6.59-6.99 (m, 1H), 7.10 (s, 1H), 8.20 (s, 2H), 8.30 (s,1H), 8.40 (br. s., 1H), 9.67 (br. s., 1H) 541.3 10

CH2

1H NMR (400 MHz, DMSO-d6) Shift: 1.54-1.69 (m, 1H), 1.91-2.05 (m, 1H),2.13-2.42 (m, 2H), 2.22 (s, 3H), 2.85 (s, 3H), 3.00-3.27 (m, 2H), 3.06(s, 3H), 3.29-3.82 (m, 2H), 3.69 (s, 2H), 4.00-4.61 (m, 2H), 4.67- 4.85(m, 1H), 5.54-5.73 (m, 1H), 6.03-6.16 (m, 1H), 6.66-6.91 (m, 1H), 7.14(d, J = 8.0 Hz, 1H), 7.34-7.41 (m, 1H), 8.16 (br s, 1H), 8.27 (s, 1H),8.49 (br s, 1H), 10.00-10.10 (m, 1H) 509.3 11

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.62 (br. s., 1H), 2.02 (br. s., 1H),2.21 (br. s., 1H), 2,29 (m, 1H), 2.84 (s, 3H), 3.06 (s, 3H), 3.79 (s,2H), 3.86 (s, 3H), 4.03 (br. s., 1H), 4.19 (br. s., 1H), 4.49 (d, J =11.62 Hz, 1H), 4.76 (br. s., 1H), 5.45-5.72 (m, 1H), 5.97-6.12 (m, 1H),6.54-6.89 (m, 1H), 7.11 (d, J = 8.20 Hz, 1H), 7.97 (d, J = 8.20 Hz, 1H),8.20 (br. s., 1H), 8.28 (s, 1H), 8.37 (br. s., 1H), 9.81 (br. s., 1H)541.3 12

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.59 (br. s., 1H), 1.94 (br. s., 1H),2.12 (s, 3H), 2.18 (br. s., 1H), 2,30 (br. s., 1H), 2.83 (s, 3H), 3.05(s, 3H), 3.10-3.25 (m, 1H), 3.70 (s, 2H), 4.05 (d, J = 15.03 Hz, 1H),3.78 (br. s., 1H), 4.13-4.30 (m, 1H), 4.55 (d, J = 11.62 Hz, 1H), 4.74(br. s., 1H), 5.54- 5.72 (m, 1H), 6.02-6.14 (m, 1H), 6.84 (br. s., 1H),7.05-7.12 (m, 1H), 7.20 (br. s., 1H), 8.09 (br. s., 1H), 8.25 (s, 1H),8.48 (br. s., 1H), 10.12-10.28 (m, 1H) 509.3 13

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.52-1.69 (m, 1H), 1.92-2.03 (m, 1H),2.06-2.41 (m, 2H), 2.10 (s, 3H), 2.13 (s, 3H), 2.85 (s, 3H), 2.99-3.24(m, 1H), 3.05 (s, 3H), 3.29-3.85 (m, 1H), 3.33 (s, 2H), 3.99-4.58 (m,2H), 4.67- 4.81 (m, 1H), 5.58-5.73 (m, 1H), 6.05-6.15 (m, 1H), 6.71-6.96(m, 1H), 6.95 (d, J = 8.0 Hz, 1H), 7.10-7.15 (m, 1H), 8.09 (br s, 1H),8.26 (s, 1H), 8.60 (br s, 1H), 10.11 -10.24 (m, 1H). 505.3 14

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.62 (br. s., 1H), 2.02 (br. s., 1H),2.21 (br. s., 1H), 2.27 (br. s., 1H), 2.83 (s, 3H), 3.05 (s, 3H), 3.71(s, 2H), 3.78 (m., 1H), 3.94 (s., 3H), 4.04 (d, J = 19.82 Hz, 2H), 4.17(br. s., 1H), 4.45 (br. s., 1H), 4.52-4.56 (m, 1H), 4.52-4.57 (m, 1H),4.71-4.87 (m, 1H), 5.38-5.77 (m, 1H), 5.98-6.11 (m, 1H), 6.51-6.90 (m,1H), 6.98 (t, J = 7.86 Hz, 1H), 7.77 (d, J = 8.20 Hz, 1H), 8.18 (br. s.,1H), 8.27 (s, 1H), 8.40 (br. s., 1H), 9.80 (br. s., 1H) 525.3 15

CH2

1H NMR (400 MHz, DMSO) Shift 1.60 (br. s., 1H), 1.96 (br. s, 1H), 2.19(br. s., 1H), 2.26 (br. s., 1H), 2.82 (s, 3H), 3.01 (s, 3H), 3.72 (s,2H), 3.95-4.18 (m, 1H), 4.19-4.54 (m, 1H), 4.77 (br. s., 1H), 5.52-5.70(m, 1H), 6.01-6.12 (m, 1H), 6.81 (d, J = 10.25 Hz, 1H), 7.12-7.21 (m,3H), 7.87 (d, J = 8.20 Hz, 1H), 8.16 (br. s., 1H), 8.27 (s, 1H), 8.39(br. s., 1H), 9.87 (br. s., 1H) 543.3 16

CH2

1H NMR (400 MHz, DMSO) Shift 1.60 (br. s., 1H), 1.96 (s, 1H), 2.19 (br.s., 2H), 2.82 (s, 3H), 2.98-3.03 (m, 3H), 3.63-3.75 (m, 2H), 3.80 (br.s., 1H), 4.00 (d, J = 7.52 Hz, 1H), 4.12-4.52 (m, 1H), 4.70-4.88 (m,1H), 5.49-5.73 (m, 1H), 5.83 (br. s., 1H), 5.94-6.12 (m, 2H), 6.59-6.91(m, 1H), 7.01 (d, J = 8.20 Hz, 1H), 7.16 (s, 1H), 7.97 (br. s., 1H),8.17 (br. s., 1H), 8.27 (s, 1H), 8.44 (br. s., 1H), 9.73 (d, J = 15.72Hz, 1H) 525.3

TABLE 3 17

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.59 (br. s., 1H), 1.96 (br. s., 1H),2.18 (br. s., 1H), 2.29 (br. s., 1H), 2.81 (s, 3H), 3.01 (s, 4H), 3.13(d, J = 5.47 Hz, 1H), 3.70 (s, 3H), 4.02 (br. s., 1H), 4.15 (br. s.,1H), 4.50 (d, J = 12.98 Hz, 1H), 4.74 (br. s., 1H), 5.51-5.69 (m, 1H),6.08 (d, J = 15.72 Hz, 1H), 6.83 (br. s., 1H), 7.26 (d, J = 8.20 Hz,1H), 7.56 (s, 1H), 7.79 (br. s., 1H), 8.09-8.19 (m, 1H), 8.26 (s, 1H),8.38 (br. s., 1H), 10.05 (s, 1H) 555.2 18

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.51-1.74 (m, 1H), 1.98 (d, J = 12.10Hz, 1H), 2.12-2.38 (m, 2H), 2.78-2.92 (m, 3H), 3.03 (s, 3H), 3.74 (s,3H), 3.98- 4.56 (m, 2H), 4.75 (br. s., 1H), 5.52-5.73 (m, 1H), 5.97-6.24(m, 1H), 6.62-6.99 (m, 1H), 7.49 (d, J = 7.33 Hz, 1H), 7.80 (d, J =10.63 Hz, 1H), 8.17-8.39 (m, 3H), 10.11 (s, 1H) 529.3 19

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.07-1.37 (m, 1H), 1.47-1.69 (m, 1H),1.88-2.03 (m, 1H), 2.11-2.30 (m, 5H), 2.83 (s, 3H), 3.05 (s, 3H), 3.74(s, 3H), 3.94-4.58 (m, 3H), 4.64-4.86 (m, 1H), 5.50-5.74 (m, 1H),5.89-6.21 (m, 1H), 6.57-6.90 (m, 1H), 7.18 (s, 1H), 7.56 (d, J = 10.93Hz, 1H), 8.12 (s, 1H), 8.25 (s, 1H), 8.45 (br. s., 1H), 10.05 (d, J =38.30 Hz, 1H) 525.3 20

CH2

No data 545.2 21

O

No data 479.3 22

CH2

1H-NMR (DMSO-D6) δ 0.56-0.71 (m, 2.0H), 0.91-1.05 (m, 2.0H), 1.54- 1.71(m, 1.0H), 1.90-2.06 (m, 2.0H), 2.16-2.34 (m, 2.0H), 2.82 (s, 3.0H),3.00 (s, 3.0H), 3.14-3.84 (m, 2.0H), 3.65 (s, 2.0H), 4.00-4.91 (m,3.0H), 5.56-5.71 (m, 1.0H), 5.96-6.18 (m, 1.0H), 6.62-6.91 (m, 1.0H),7.00 (s, 1.0H), 7.06-7.12 (m, 1.0H), 7.74- 7.81 (m, 1.0H), 8.21 (s,1.0H), 8.28 (s, 1.0H), 8.57 (s, 1.0H), 9.98-10.11 (m, 1.0H). 517.3 23

CH2

1H-NMR (DMSO-D6) δ: 1.49-1.69 (m, 1.0H), 1.86-2.03 (m, 1.0H), 2.16- 2.53(m, 2.0H), 2.34-3.85 (m, 2.0H), 2.84 (s, 3.0H), 3.04 (s, 3.0H), 3.80 (s,2.0H), 4.04-4.87 (m, 3.0H), 5.60-5.78 (m, 1.0H), 6.01-6.20 (m, 1.0H),6.73- 6.92 (m, 1.0H), 7.54-7.64 (m, 2.0H), 7.72 (s, 1.0H), 8.18 (s,1.0H), 8.29 (s, 1.0H), 8.37 (s, 1.0H), 10.79 (s, 1.0H). 502.3 24

CH2

1H-NMR (DMSO-D6) δ: 1.58-1.67 (m, 1.0H), 1.93-2.04 (m, 1.0H), 2.12- 2.46(m, 5.0H), 2.85 (s, 3.0H), 2.96- 3.81 (m, 2.0H), 3.09 (s, 3.0H), 3.89(s, 2.0H), 4.06-4.80 (m, 3.0H), 5.56- 5.74 (m, 1.0H), 6.04-6.16 (m,1.0H), 6.67-6.94 (m, 1.0H), 7.36 (s, 1.0H), 7.85-7.91 (m, 1.0H), 8.16(s, 1.0H), 8.27 (s, 1.0H), 8.46 (s, 1.0H), 10.15- 10.38 (m, 1.0H). 516.325

CH2

1H-NMR (DMSO-D6) δ: 1.56-1.63 (m, 1.0H), 1.92-1.96 (m, 1.0H), 2.14- 2.36(m, 2.0H), 2.82 (s, 3.0H), 2.88- 3.75 (m, 2.0H), 3.02 (s, 3.0H), 3.72(s, 2.0H), 4.00-4.80 (m, 3.0H), 5.54-5.74 (m, 1.0H), 6.01-6.14 (m,1.0H), 6.70- 6.88 (m, 1.0H), 7.07 (dd, J = 8.2, 1.3 Hz, 1.0H), 7.16 (dd,J = 11.7, 1.3 Hz, 1.0H), 7.53-7.60 (m, 1.0H), 8.12 (s, 1.0H), 8.26 (s,1.0H), 8.43 (s, 1.0H), 10.20-10.15 (m, 1.0H). 495.3

TABLE 4 26

O

1H NMR (400 MHz, DMSO-d6) Shift 0.03 (br. s., 1H), 1.57 (br. s., 1H),1.96 (br. s., 1H), 2.22 (br. s., 3H), 2.26-2.29 (m, 1H), 2.88 (br. s.,3H), 3.01 (br. s., 3H), 3.78 (br. s., 1H), 4.03 (br. s., 1H), 4.14 (br.s., 1H), 4.21 (br. s., 1H), 4.50 (br. s., 1H), 4.73 (br. s., 1H),5.52-5.73 (m, 1H), 5.58 (br. s., 1H), 5.68 (br. s., 1H), 5.97-6.15 (m,1H), 6.06 (s, 1H), 6.10 (s, 1H), 6.64-6.89 (m, 1H), 6.69 (br. s., 1H),6.83 (br. s., 1H), 6.96 (br. s., 1H), 7.03 (br. s., 1H), 7.40 (br. s.,1H), 8.06 (br. s., 493.3 1H), 8.24 (br. s., 1H), 8.51 (br. s., 1H),9.98-10.14 (m, 1H), 10.01 (br. s., 1H), 10.09 (br. s., 1H) 27

O

1H NMR (400 MHz, DMSO-d6) Shift 1.59 (br. s., 1H), 1.94 (br. s., 1H),2.18 (br. s., 1H), 2.29 (br. s., 1H), 2.89 (s, 3H), 3.02 (s, 3H), 3.74(br. s., 1H), 4.04 (br. s., 1H), 4.54 (br. s., 1H), 4.74 (br. s., 1H),5.53-5.70 (m, 1H), 6.08 (d, J = 14.35 Hz, 1H), 6.62-6.88 (m, 1H), 7.17(dd, J = 8.54, 2.39 Hz, 1H), 7.42 (d, J = 2.73 Hz, 1H), 7.79 (d, J =8.88 Hz, 1H), 8.13 (br. s., 1H), 8.26 (s, 1H), 8.38 (br. s., 1H), 10.16(br. s., 1H) 513.2 28

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.59 (br. s., 1H), 1.90 (br. s., 1H),2.15 (br. s., 1H), 2.29 (br. s., 1H), 2.81 (d, J = 4.10 Hz, 3H), 2.99(d, J = 4.10 Hz, 4H), 3.64 (br. s., 2H), 3.82 (br. s., 1H), 3.96-4.14(m, 1H), 4.14-4.59 (m, 1H), 4.63-4.86 (m, 1H), 5.53- 5.79 (m, 1H), 6.09(br. s., 1H), 6.65-6.92 (m, 1H), 7.21 (br. s., 2H), 7.71 (br. s., 2H),8.10 (br. s., 1H), 8.22 (d, J = 15.03 Hz, 1H), 8.49 (br. s., 1H), 10.09-10.38 (m, 1H) 477.3 29

O

1H NMR (400 MHz, DMSO-d6) Shift 1.57 (br. s., 1H), 1.90 (br. s., 1H),2.15 (br. s., 1H), 2.29 (br. s., 1H), 2.90 (br. s., 3H), 3.05 (br. s.,4H), 3.79 (br. s., 1H), 4.06 (br. s., 1H), 4.22 (br. s., 1H), 4.53 (br.s., 1H), 4.70 (br. s., 1H), 5.55-5.71 (m, 1H), 6.09 (br. s., 1H), 6.85(br. s., 1H), 7.31 (br. s., 1H), 7.78 (d, J = 6.83 Hz, 1H), 8.04 (br.s., 1H), 8.14 (br. s., 1H), 8.25 (br. s., 1H), 8.37 (br. s., 1H), 8.43(br. s., 1H), 10.32-10.67 (m, 1H) 513.2 30

O

1H NMR (400 MHz, DMSO-d6) Shift 1.58 (br. s., 1H), 1.90 (br. s., 1H),2.12 (br. s., 4H), 2.28 (br. s., 1H), 2.89 (br. s., 3H), 3.05 (br. s.,3H), 3.75-3.91 (m, 1H), 3.97-4.26 (m, 2H), 4.42- 4.58 (m, 1H), 4.63-4.85(m, 1H), 5.51-5.78 (m, 1H), 6.09 (br. s., 1H), 6.63-6.93 (m, 1H), 7.05(br. s., 1H), 7.61 (br. s., 1H), 7.69 (br. s., 1H), 8.11 (br. s., 1H),8.24 (br. s., 1H), 8.36 (br. s., 1H), 8.47 (br. s., 1H), 10.03-10.46 (m,1H) 493.3 31

O

1H NMR (400 MHz, DMSO-d6) Shift 1.58 (br. s., 1H), 1.91 (br. s., 1H),2.16 (br. s., 1H), 2.30 (br. s., 1H), 2.89 (br. s., 4H), 3.02 (br. s.,3H), 3.72 (br. s., 1H), 4.07 (br. s., 1H), 4.25 (br. s., 1H), 4.55 (br.s., 1H), 4.73 (br. s., 1H), 5.56-5.71 (m, 1H), 6.11 (br. s., 1H),6.66-6.88 (m, 1H), 7.01 (d, J = 6.15 Hz, 1H), 7.20 (d, J = 8.88 Hz, 1H),7.60 (br. s., 1H), 8.10 (br. s., 1H), 8.25 (br. s., 1H), 8.43 (br. s.,1H), 10.26 (br. s., 1H) 497.3 32

O

1H NMR (400 MHz, DMSO-d6) Shift 1.57 (br. s., 1H), 1.94 (br. s., 1H),2.05 (br. s., 3H), 2.12 (br. s., 4H), 2.30 (br. s., 1H), 2.89 (br. s.,3H), 3.06 (br. s., 3H), 3.76 (br. s., 1H), 4.02 (br. s., 1H), 4.18 (br.s., 1H), 4.53 (br. s., 1H), 4.74 (br. s., 1H), 5.51-5.70 (m, 1H), 6.07(br. s., 1H), 6.63-6.88 (m, 1H), 6.91 (d, J = 7.52 Hz, 1H), 7,19 (br.s., 1H), 8.05 (br. s., 1H), 8.24 (br. s., 1H), 8.54 (br. s., 1H),10.08-10.25 (m, 1H) 509.3 33

O

1H NNR (400 MHz, DMSO-d6) Shift 1.60 (br. s., 1H), 1.96 (br. s., 1H),2.21 (br. s., 1H), 2.96 (br. s., 3H), 3.14 (s, 3H), 3.76-3.91 (m, 1H),3.97-4.39 (m, 2H), 4.49- 4.64 (m, 1H), 4.75 (br. s., 1H), 5.50-5.80 (m,1H), 6.10 (d, J = 16.40 Hz, 1H), 6.60-6.95 (m, 1H), 7.32 (d, J = 7.52Hz, 1H), 7.60 (br. s., 4H), 7.93 (br. s., 3H), 8.05 (br, s., 1H), 8.26(br, s., 1H), 8.36 (br. s., 1H), 8.47 (br. s., 1H), 10.57-10.83 (m, 1H)529.3

TABLE 5 34

O

1H NMR (400 MHz, DMSO-d6) Shift 1.57 (br. s., 1H), 1.90 (br. s., 1H),2.15 (br. s., 1H), 2.29 (br. s., 1H), 2.89 (s, 3H), 3.03 (s, 4H), 3.81(br. s., 1H), 4.05 (d, J = 12.98 Hz, 1H), 4.12- 4.27 (m, 1H), 4.54 (d, J= 10.93 Hz, 1H), 4.70 (br. s., 1H), 5.55-5.71 (m, 1H), 6.10 (d, J =14.35 Hz, 1H), 6.64- 6.88 (m, 1H), 7.27 (t, J = 8.54 Hz, 1H), 7.62 (d, J= 8.88 Hz, 1H), 7.85 (d, J = 12.30 Hz, 1H), 8.14 (br. s., 1H), 8.25 (s,1H), 8.40 (d, J = 16.40 Hz, 1H), 10.35-10.59 (m, 1H) 497.3 35

O

1H NMR (400 MHz, DMSO-d6) Shift 1.58 (br. s., 1H), 1.92 (br. s., 1H),2.17 (br. s., 1H), 2.30 (br. s., 1H), 2.50 (s, 2H), 2.91 (s, 4H), 3.05(s, 4H), 3.71 (br. s., 1H), 4.05 (br. s., 1H), 4.22-4.64 (m, 2H), 4.71(br. s., 1H), 5.64 (d, J = 17.77 Hz, 1H), 6.09 (d, J = 17.77 Hz, 1H),6.64-6.91 (m, 1H), 7.14-7.21 (m, 1H), 7.40 (br. s., 1H), 8.11 (br. s.,1H), 8.25 (s, 1H), 8.41 (s, 1H), 10.33-10.67 (m, 1H) 515.3 36

O

1H NMR (400 MHz, DMSO-d6) Shift 1.21 (br. s., 1H), 1.58 (m, 1H), 1.96(br. s., 1H), 2.12-2.35 (m, 2H), 2.89 (s, 3H), 3.02 (s, 3H), 3.79 (br.s., 1H), 3.84-3.90 (m, 3H), 3.99 (d, J = 11.62 Hz, 1H), 4.45 (d, J =12.30 Hz, 0.5H), 4.19 (d, J = 12.98 Hz, 0.5H), 4.76 (br. s., 1H), 5.65(d, J = 10.25 Hz, 1H), 5.99-6.10 (m, 1H), 6.58-6.88 (m, 2H), 6.92 (d, J= 2.73 Hz, 1H), 8.01 (d, J = 8.88 Hz, 1H), 8.15 (br. s., 1H), 8.22- 8.28(m, 1H), 8.46 (br. s., 1H), 9.62 (br. s., 1H) 509.3 37

O

1H NMR (400 MHz, DMSO-d6) Shift 1.66 (br. s., 1H), 1.97-2.17 (m, 1H),2.25 (br. s., 1H), 2.28-2.38 (m, 1H), 2.94 (s, 3H), 3.12-3.22 (m, 5H),3.76- 4.17 (m, 2H), 4.20-4.51 (m, 1H), 4.80 (br. s., 1H), 5.51-5.61 (m,1H), 6.04 (d, J = 15.03 Hz, 1H), 6.61-6.88 (m, 1H), 7.44 (d, J = 8.88Hz, 1H), 7.72 (dd, J = 8.54, 4.44 Hz, 1H), 8.24 (s, 1H), 8.29 (s, 1H),8.37 (d, J = 8.20 Hz, 1H), 8.50 (br. s., 1H), 8.72 (d, J = 8.88 Hz, 1H),9.05 (d, J = 3.42 Hz, 1H), 11.34 (br. s., 1H) 530.3 38

O

1H NMR (400 MHz, DMSO-d6) Shift 1.57 (br. s., 1H), 1.94 (br. s., 1H),2.08 (s, 3H), 2.18 (s, 4H), 2.29 (br. s., 1H), 2.89 (s, 3H), 3.05 (s,3H), 3.77 (br. s., 1H), 3.97-4.35 (m, 2H), 4.52 (d, J = 10.93 Hz, 1H),4.71 (br. s., 1H), 5.52-5.70 (m, 1H), 6.00-6.12 (m, 1H), 6.60-6.88 (m,1H), 6.96 (s, 1H), 7.30 (br. s., 1H), 8.06 (br. s., 1H), 8.24 (s, 1H),8.52 (br. s., 1H), 9.91- 10.09 (m, 1H) 507.3 39

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.58 (br. s., 1H), 1.96 (br. s., 1H),2.13 (s, 3H), 2.16 (s, 2H), 2.23 (s, 1H), 2.29 (br s., 1H), 2.83 (s,3H), 3.02 (s, 3H), 3.61 (s, 2H), 3.77 (br. s., 1H), 3.97-4.36 (m, 2H),4.49 (br. s., 1H), 4.77 (br. s., 1H), 5.50-5.70 (m, 1H), 5.99-6.12 (m,1H), 6.83 (s, 1H), 6.94 (s, 1H), 7.23 (br. s., 1H), 8.10 (br. s., 1H),8.24 (s, 1H), 8.57 (br. s., 1H), 9.83-10.02 (m, 1H) 505.3 40

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.57 (br. s., 1H), 1.87 (d, J = 19.13Hz, 1H), 2.15 (br. s., 1H), 2.29 (br. s., 1H), 2.80 (s, 3H), 2.98 (s,3H), 3.76 (s, 3H), 3.84 (br. s., 1H), 3.99-4.30 (m, 3H), 4.51 (br. s.,1H), 4.70 (br. s., 1H), 5.56-5.71 (m, 1H), 6.10 (d, J = 13.67 Hz, 1H),6.82 (d, J = 8.88 Hz, 1H), 7.02-7.07 (m, J = 8.20 Hz, 1H), 7.36-7.42 (m,J = 8.20 Hz, 1H), 7.47 (br. s., 1H), 8.08-8.19 (m, 2H), 8.25 (s, 1H),8.49 (br. s., 1H), 10.06- 10.30 (m, 1H) 507.3 41

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.66 (br. s., 1H), 2.00-2.17 (m, 1H),2.25 (br. s., 1H), 2.29-2.44 (m, 1H), 2.83 (s, 3H), 3.09-3.17 (m, 3H),3.33- 3.55 (m, 2H), 4.14 (s, 3H), 4.21-4.51 (m, 1H), 4.81 (br. s., 1H),5.51-5.82 (m, 1H), 6.04 (d, J = 16.40 Hz, 1H), 6.82 (d, J = 10.93 Hz,1H), 7.45 (d, J = 8.20 Hz, 1H), 7.66 (dd, J = 8.88, 4.10 Hz, 1H), 8.24(s, 1H), 8.29 (s, 1H), 8.43 (d, J = 8.88 Hz, 1H), 8.54 (br. s., 1H),8.66 (d, J = 8.20 Hz, 1H), 8.99 (d, J = 4.10 Hz, 1H), 11.46 (br. s., 1H)528.3

TABLE 6 42

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.56 (br. s., 1H), 1.84 (br. s., 1H),2.14 (br. s., 1H), 2.29 (br. s., 1H), 2.81 (s, 3H), 3.02 (s, 3H), 3.66(s, 2H), 3.82 (br. s., 1H), 4.09 (br. s., 2H), 4.44-4.56 (m, 1M), 4.60-4.85 (m, 1H), 5.49-5.75 (m, 1H), 6.10 (d, J = 13.67 Hz, 1H), 6.60- 6.95(m, 1H), 7.22 (t, J = 8.20 Hz, 1H), 7.55 (d, J = 8.20 Hz, 1H), 7.69 (d,J = 11.62 Hz, 1H), 8.11 (s, 1H), 8.25 (s, 1H), 8.40 (br. s., 1H),10.26-10.57 (m, 1H) 495.3 43

O

1H NMR (400 MHz, DMSO-d6) Shift 1.58 (br. s., 1H), 1.91 (br. s., 1H),2.16 (br. s., 1H), 2.21-2.44 (m, 1H). 2.92 (s, 3H), 3.06 (s, 3H), 3.79(br. s., 1H), 4.07 (d, J = 13.67 Hz, 1H), 4.24 (br. s., 1H), 4.57 (d, J= 12.98 Hz, 1H), 4.71 (br. s., 1H), 5.58-5.71 (m, 1H), 6.10 (d, J =15.03 Hz, 1H), 6.78-6.89 (m, 1H), 7.75 (d, J = 9.57 Hz, 2H), 8.18 (br.s., 1H), 8.26 (br. s., 2H), 10.53- 10.72 (m, 1H) 515.3 44

O

1H NMR (400 MHz, DMSO-d6) Shift 1.57 (br. s., 1H), 1.95 (br. s., 1H),2.15 (s, 7H), 2.30 (br. s., 1H), 2.88 (s, 3H), 3.01 (s, 3H), 3.78 (m.,2H), 4.04 m., 2H), 4.17 (m., 2H), 4.51 (m, 1H), 4.71 (br. s., 1H), 5.66(br. s., 1H), 6.08 (d, J = 15.72 Hz, 1H), 6.64-6.85 (m, 1H), 6.87 (s,2H), 8.02 (br. s., 1H), 8.21 (d, J = 17.08 Hz, 2H), 8.55 (br. s., 1H),9.94-10.15 (m, 1H) 507.3 45

O

1H NMR (400 MHz, DMSO-d6) Shift 1.58 (br. s., 1H), 1.89 (d, J = 10.93Hz, 1H), 2.16 (br. s., 1H), 2.22-2.43 (m, 1H), 2.92 (s, 3H), 3.09 (s,3H), 3.77 (br. s., 1H), 4.08 (br. s., 1H), 4.24 (br. s., 1H), 4.54 (br.s., 1H), 4.70 (br. s., 1H), 5.54- 5.71 (m, 1H), 6.04-6.14 (m, 1H),6.59-6.96 (m, 1H), 8.05 (s, 2H), 8.16 (br. s., 1H), 8.26 (br. s., 2H),10.64 (br. s., 1H) 547.2 46

O

1H NMR (400 MHz, DMSO-d6) Shift 1.57 (br. s., 1H), 1.87 (d, J = 18.45Hz, 1H), 2.09 (s, 7H), 2.19- 2.41 (m, 1H), 2.90 (s, 3H), 3.08 (s, 3H),3.76-3.89 (m, 1H), 4.06 (d, J = 4.78 Hz, 3H), 4.17 (br. s., 1H),4.43-4.61 (m, 1H), 4.64-4.87 (m, 1H), 5.68 (d, J = 10.25 Hz, 1H), 6.11(d, J = 15.03 Hz, 1H), 6.63-6.95 (m, 1H), 7.52 (s, 2H), 8.12 (br. s.,1H), 8.25 (s, 1H), 8.48 (br. s., 1H), 10.04-10.27 (m, 1H) 507.3 47

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.56-1.81 (m, 1H), 2.08 (br. s., 1H),2.24 (d, J = 4.78 Hz, 2H), 2.69 (s, 6H), 2.84 (s, 3H), 3.02 (s, 3H),3.67 (s, 2H), 3.95 (br. s., 2H), 4.08 (br. s., 2H), 4.31-4.50 (m, 1H),4.71-4.96 (m, 1H), 5.40-5.75 (m, 1H), 5.89-6.15 (m, 1H), 6.47-6.93 (m,1H), 7.00 (d, J = 8.20 Hz, 1H), 7.17 (s, 1H), 8.12-8.24 (m, 3H), 8.30(s, 1H), 8.52 (br. s., 1H), 10.08 (br. s., 1H) 520.3 48

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.58 (br. s., 1H), 1.93 (br. s., 1H),2.17 (br. s., 1H), 2.29 (br. s., 2H), 2.50 (s, 1H), 2.82 (s, 3H), 3.02(s, 4H), 3.70 (s, 4H), 4.07 (q, J = 5.01 Hz, 2H), 4.12-4.31 (m, 1H),4.46-4.81 (m, 2H), 5.31 (d, J = 11.62 Hz, 1H), 5.53-5.67 (m, 1H), 5.75(d, J = 17.08 Hz, 1H), 6.07 (br. s., 1H), 6.80 (dd, J = 17.43, 11.28 Hz,2H), 7.16 (d, J = 6.15 Hz, 1H), 7.33 (d, J = 8.20 Hz, 1H), 7.50 (s, 1H),8.04  

503.3 49

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.11 (t, J = 7.52 Hz, 3H), 1.58 (br. s.,1H), 1.94 (br. s., 1H), 2.17 (br. s., 1H), 2.29 (br. s., 1H), 2.59 (q, J= 7.52 Hz, 2H), 2.81 (s, 3H), 2.99 (s, 3H), 3.66 (s, 2H), 3.74-4.11 (m,1H), 4.14-4.57 (m, 1H), 4.72 (br. s., 1H), 5.50-5.75 (m, 1H), 5.99-6.12(m, 1H), 6.84 (br. s., 1H), 7.06 (d, J = 8.20 Hz, 1H), 7.13 (s, 1H),7.35 (br. s., 1H), 7.98-8.12 (m, 1H), 8.24 (s, 1H), 8.52 (br. s., 1H),9.86-10.07 (m, 1H) 505.3

indicates data missing or illegible when filed

TABLE 7 50

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.15-1.36 (m, 1H), 1.45- 1.69 (m, 1H),1.86-2.04 (m, 1H), 2.15-2.37 (m, 2H), 2.81 (s, 3H), 3.05 (s, 3H), 3.78(s, 2H), 3.94- 4.61 (m, 3H), 4.64-4.88 (m, 1H), 5.54-5.75 (m, 1H),5.97-6.17 (m, 1H),6.60-6.93 (m, 1H), 7.26 (t, J = 7.93 Hz, 1H), 7.60 (d,J = 8.25 Hz, 1H), 8.07-8.46 (m, 3H), 10.24 (s, 1H) 529.3 51

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.60 (br. s., 1H), 1.96 (br. s., 1H),2.15-2.32 (m, 2H), 2.82 (s, 3H), 3.02 (s, 3H), 3.73 (s, 3H), 4.07 (br.s., 2H), 4.17 (br. s., 1H), 4.23-4.62 (m, 1H), 4.74 (br. s., 1H),5.49-5.70 (m, 1H), 5.99-6.12 (m, 1H), 6.64 (br. s., 1H), 6.65-6.83 (m,1H), 6.88 (d, J = 2.73 Hz, 1H), 7.22 (d, J = 8.20 Hz, 1H), 7.60 (s, 1H),7.84 (s, 1H), 7.89 (br. s., 1H), 8.08- 8.17 (m, 2H), 8.26 (s, 1H), 8.42(br. s., 1H), 10.32 (d, J = 12.98 Hz, 1H) 543.3 52

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.57 (br. s., 1H), 1.94 (d, J = 14.35Hz, 1H), 2.08-2.28 (m, 2H), 2.82 (s, 3H), 3.02 (s, 3H), 3.71 (s, 2H),4.05 (br. s., 1H), 4.50 (d, J = 11.62 Hz, 1H), 4.71 (br. s., 1H),5.51-5.69 (m, 1H), 6.08 (d, J = 17.08 Hz, 1H), 6.82 (s, 2H), 7.20 (d, J= 8.20 Hz, 1H), 7.36 (s, 1H), 7.80 (br. s., 2H), 8.00 (s, 1H), 8.11 (br.s., 1H), 8.25 (s, 1H), 8.40 (br. s., 1H), 9.92 (br. s., 1H) 543.3 53

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.08-1.35 (m, 1H), 1.49- 1.71 (m, 1H),1.86-2.02 (m, 1H), 2.08-2.38 (m, 2H), 2.81 (s, 3H), 3.01 (s, 3H), 3.60(s, 2H), 3.68- 4.61 (m, 3H), 4.64-4.90 (m, 1H), 5.58-5.81 (m, 1H), 6.01(s, 2H), 6.09 (s, 1H), 6.68 (d, J = 8.20 Hz, 1H), 6.71-6.89 (m, 1H),6.97 (d, J = 7.52 Hz, 1H), 8.02-8.15 (m, 1H), 8.24 (s, 1H), 8.46 (br.s., 1H), 10.10 (br. s., 1H) 521.3 54

CH2

1H NMR (400 MHz, DMSO-d6) Shift 2.37-2.43 (m, 1H), 2.01 (s, 3H), 2.99(s, 3H), 3.56-3.65 (m, 1H), 3.69 (s, 2H), 3.73-3.85 (m, 1H), 3.85-4.01(m, 2H), 4.02-4.16 (m, 1H), 5.44-5.59 (m, 1H), 5.60- 5.72 (m, 1H),6.07-6.19 (m, 1H), 6.50-6.70 (m, 1H), 7.16-7.26 (m, 1H), 7.36-7.44 (m,1H), 7.82- 7.95 (m, 1H), 8.09-8.20 (m, 1H), 8.23-8.28 (m, 1H), 8.30-8.43(m, 1H), 9.88-10.10 (m, 1H) 497.3 55

CH2

1H NMR (400 MHz, DMSO-d6) Shift 2.83 (s, 3H), 3.05 (s, 3H), 3.59-3.81(m, 7H), 3.31-3.99 (m, 3H), 4.00-4.18 (m, 1H), 5.43- 5.76 (m, 2H),6.09-6.24 (m, 1H), 6.48-6.74 (m, 1H), 7.05-7.12 (m, 1H), 7.95-8.07 (m,1H), 8.15- 8.25 (m, 1H), 8.25-8.33 (m, 1H), 8.33-8.44 (m, 1H), 9.70-9.78(m, 1H) 527.3 56

CH2

1H NMR (400 MHz, DMSO-d6) Shift 2.09 (s, 3H), 2.41 (br. s., 1H), 2.83(s, 3H), 3.04 (s, 3H), 3.61 (br. s., 1H), 3.69 (s, 2H), 3.74-3.87 (m,1H), 3.91 (br. s., 1H), 3.98-4.17 (m, 2H), 5.42- 5.59 (m, 1H), 5.66 (dd,J = 17.08, 10.25 Hz, 1H), 6.14 (d, J = 16.40 Hz, 1H), 6.52-6.68 (m, 1H),7.05-7.12 (m, 1H), 7.23 (t, J = 7.86 Hz, 1H), 8.14 (br. s., 1H), 8.27(s, 1H), 8.53 (br. s., 1H), 10.18 (d, J = 15.72 Hz, 1H) 495.3 57

CH2

1H NMR (400 MHz, DMSO-d6) Shift 2.39 (br. s., 1H), 2.82 (s, 3H), 3.04(s, 3H), 3.70 (s, 2H), 3.75-3.96 (m, 6H), 4.02-4.14 (m, 1H), 5.45-5.73(m, 2H), 5.16 (dd, J = 17.08, 8.20 Hz, 1H), 6.53- 6.71 (m, 1H), 6.97 (t,J = 8.20 Hz, 1H), 7.82 (d, J = 4.78 Hz, 1H), 8.21 (br. s., 1H), 8.29 (s,1H), 8.42 (br. s., 1H), 9.74 (br. s., 1H) 511.2 58

O

1H NMR (400 MHz, DMSO-46) Shift 2.50-2.67 (m, 2H), 2.96 (s, 3H), 3.14(s, 3H), 3.63 (br. s., 1H), 3.80 (d, J = 5.47 Hz, 1H), 3.86-4.04 (m,2H), 4.04-4.20 (m, 2H), 5.51-5.71 (m, 2H), 6.11-6.19 (m, 1H), 6.56-6.70(m, 1H), 7.33 (d, J = 3.20 Hz, 1H), 7.57-7.66 (m, 3H), 7.91 (dd, J =6.15, 2.73 Hz, 2H), 8.07 (br. s., 1H), 8 24- 8.31 (m, 1H), 8.48 (br. s.,1H), 10.68 (d, J = 6.53 Hz, 1H) 515.3

TABLE 8 59

CH2

1H NMR (400 MHz, DMSO-d6) Shift 2.11 (s, 3H), 2.12 (s, 3H), 2.35- 2.70(m, 2H), 2.86 (s, 3H), 3.05 (s, 3H), 3.47- 4.25 (m, 4H), 3.71 (s, 2H),5.48-5.61 (m, 1H), 5.62-5.75 (m, 1H), 6.11- 6.19 (m, 1H), 6.52-6.72 (m,1H), 6.96 (d, J = 8.2 Hz, 1H), 7.12-7.22 (m, 1H), 8.08 (br. s., 1H),8.27 (s., 1H), 8.59 (br. s., 1H), 10.10-10.30 (m, 1H) 491.5 60

CH2

1H NMR (400 MHz, DMSO-d6) Shift 2.81 (s, 3H), 3.01 (s, 3H), 3.61 (br.s., 2H), 3.70 (s, 2H), 3.75-3.96 (m, 2H), 4.00- 4.24 (m, 2H), 5.24-5.80(m, 3H), 6.13 (d, J = 15.03 Hz, 1H), 6.50-6.67 (m, 1H), 6.78 (dd, J =17.77, 10.93 Hz, 1H), 7.16 (d, J = 8.20 Hz, 1H), 7.37 (d, J = 8.20 Hz,1H), 7.49 (s, 1H), 8.08 (br. s., 1H), 8.25 (s, 1H), 8.50 489.3 (br. s.,1H), 10.20 (d, J = 11.62 Hz, 1H) 61

CH2

1H NMR (400 MHz, DMSO-d6) Shift 2.49- 2.64 (m, 2H), 2.85 (s, 3H), 3.07(s, 3H), 3.61 (br. s., 1H), 3.79 (m, 1H), 3.85-4.02 (m, 2H), 4.03- 4.11(m, 2H), 4.15 (s, 2H), 5.49-5.69 (m, 2H), 6.14 (dd, J = 16.74, 2.39 Hz,1H), 6.55-6.69 (m, 1H), 7.35 (d, J = 7.52 Hz, 1H), 7.49-7.64 (m, 3H),7.87-7.39 (m, 2H), 8.01-8.15 (m, 1H), 8.26 513.3 (s, 1H), 8.50 (br. s.,1H), 10.62 (d, J = 6.83 Hz, 1H) 62

CH2

1H NMR (400 MHz, DMSO-d6) Shift 4.81 (s, 8H), 3.00 (s, 3H), 3.61 (br.s., 1H), 3.69 (s, 2H), 3.81 (d, J = 8.88 Hz, 1H), 3.90 (br. s., 1H),3.97 (d, J = 8.20 Hz, 1H), 4.03- 4.17 (m, 2H), 5.56-5.70 (m, 2H),6.06-6.22 (m, 1H), 6.53-6.69 (m, 1H), 7.25 (d, J = 8.20 Hz, 1H),7.50-7.58 (m, 1H), 7.93 (dd, J = 8.20, 4.10 Hz, 1H), 8.17 (br. s., 1H),541.2 8.27 (s, 1H), 8.34 (br. s., 1H), 9.87-10.05 (m, 1H) 63

O

1H NMR (400 MHz, DMSO-d6) Shift 2.16- 2.23 (m, 3H), 2.31-2.42 (m, 1H),2.88 (s, 3H), 3.01 (s, 3H), 3.58 (br. s., 1H), 3.68-3.94 (m, 2H), 3.99-4.15 (m, 2H), 5.41-5.69 (m, 2H), 6.09-6.16 (m, 1H), 6.52-6.67 (m, 1H),6.96 (dd, J = 8.54, 2.39 Hz, 1H), 7.03 (br. s., 1H), 7.44 (dd, J = 8.88,5.47 Hz, 1H), 8.08 (br. s., 1H), 8.25 (s, 1H), 8.51 (br. s., 479.3 1H),10.01 (d, J = 12.98 Hz, 1H) 64

O

1H NMR (400 MHz, DMSO-d6) Shift 2.05 (s, 3H), 2.10 (s, 3H), 2.52 (dd, J= 13.67, 6.15 Hz, 1H), 2.65-2.95 (m, 3H), 3.06 (s, 3H), 3.49-3.63 (m,1H), 3.70-3.95 (m, 2H), 3.99-4.16 (m, 2H), 5.45-5.69 (m, 2H), 6.08- 6.16(m, 1H), 6.52-6.67 (m, 1H), 6.91 (d, J = 8.88 Hz, 1H), 7.21 (dd, J =8.54, 4.44 Hz, 1H), 8.06 (br. s., 1H), 8.24 (s, 1H), 493.3 8.53 (br. s.,1H), 10.15 (d, J = 13.67 Hz, 1H) 65

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.10- 1.29 (m, 1H), 2.82 (s, 3H), 3.02(s, 3H), 3.73- 3.88 (m, 3H), 3.88-4.24 (m, 3H), 5.43-5.76 (m, 2H), 6.14(dd, J = 16.50, 6.35 Hz, 1H), 6.47-6.72 (m, 1H), 7.26 (t, J = 7.93 Hz,1H), 7.70 (t, J = 9.20 Hz, 1H), 8.09-8.37 (m, 3H), 8.46 (s, 1H), 10.02-10.31 (m, 1H) 515.3 66

O

1H NMR (400 MHz, DMSO-d6) Shift 1.56- 1.72 (m, 1H), 1.94-2.09 (m, 1H),2.26 (br. s., 1H), 2.53-2.62 (m, 8H), 2.99 (s, 3H), 3.24 (s., 3H),3.80-3.97 (m, 1H), 3.97-4.12 (m, 1H), 4.17- 4.69 (m, 1H), 4.71-4.94 (m,1H), 6.51-6.69 (m, 1H), 6.71-6.99 (m, 1H), 7.31-7.43 (m, 1H), 7.65 (br.s., 4H), 7.92-8.01 (m, 2H), 8.04-8.17 (m, 1H), 8.25-8.38 (m, 1H), 8.44-8.58 (m, 1H), 10.65-10.79 583.4 (m, 1H) 67

CH2

1H NMR (400 MHz, DMSO-d6) Shift 1.16- 1.27 (m, 1H), 2.18 (s, 3H), 2.82(s, 3H), 3.04 (s, 3H), 3.40-3.65 (m, 4H), 3.68-3.96 (m, 4H), 4.00- 4.36(m, 2H), 5.46-5.74 (m, 2H), 6.13 (dd, J = 16.40, 5.47 Hz, 1H), 6.49-6.82(m, 1H), 7.17 (s, 1H), 7.61 (d, J = 6.15 Hz, 1H), 8.11 (br. s., 1H),8.26 (s, 1H), 8.44 (br. s., 1H), 9.99 (d, J = 12.30 511.2 Hz, 1H)

TABLE 9 68

O

1H NMR (400 MHz, DMSO- d6) Shift 1.57 (br. s., 1H), 1.93 (br. s., 1H),2.12-2.26 (m, 4H), 2.29 (br. s., 1H), 3.77 (br. s., 1H), 3.96-4.18 (m,1H), 4.18-4.57 (m, 1H), 4.71 (br. s., 1H), 5.52-5.70 (m, 1H), 6.01-6.12(m, 1H), 6.62-6.85 (m, 1H), 7.00 (br. s., 1H), 7.08 (br. s., 1H), 7.25(br. s., 1H), 7.37-7.50 (m, 5H), 8.07 (br. s., 1H), 8.24 (br. s., 1H),8.50 (br. s., 1H), 9.97-10.14 (m, 1H) 555.3 69

O

1H NMR (400 MHz, DMSO- d6) Shift 1.58 (br. s., 1H), 1.86 (br s., 6H),2.12-2.26 (m, 4H), 2.30 (br. s., 1H), 3.77 (br. s., 1H), 3.97-4.17 (m,1H), 4.20-4.57 (m, 1H), 4.71 (br. s., 1H), 5.53-5.70 (m, 1H), 6.00-6.13(m, 1H), 6.62- 6.90 (m, 1H), 6.98 (br. s., 1H), 7.05 (br. s., 1H), 7.41(br. s., 1H), 8.06 (br. s., 1H), 8.24 (br s., 1H), 8.52 (br. s., 1H),9.96-10.19 (m, 1H) 519.3 70

O

1H NMR (400 MHz, DMSO- d6) Shift 1.54 (d, J = 12.30 Hz, 8H), 1.95 (br.s., 1H), 2.17 (br. s., 1H), 2.22 (br. s., 3H), 2.30 (br. s., 1H), 3.52(br. s., 4H), 3.77 (br. s., 1H), 3.97-4.17 (m, 1H), 4.19-4.56 (m, 1H),4.73 (br. s., 1H), 5.52-5.70 (m, 1H), 6.01-6.12 (m, 1H), 6.59-6.80 (m,1H), 6.97 (br. s., 1H), 7.04 (br. s., 1H), 7.40 (br. s., 1H), 8.07 (br.s., 1H), 8.24 (br. s., 1H), 8.52 (br. s., 1H), 9.95-10.13 (m, 1H) 533.371

CH2

1H NMR (400 MHz, DMSO- d6) Shift 1.20 (br. s., 1H), 1.58 (br. s., 1H),1.74 (d, J = 5.47 Hz, 2H), 1.80-1.90 (m, 2H), 1.95 (br. s., 1H), 2.21(br. s., 5H), 2.30 (br. s., 1H), 3.57 (br. s., 3H), 3.79 (br. s., 1H),3.97-4.14 (m, 2H), 4.15-4.57 (m, 1H), 4.70 (br. s., 1H), 5.51-5.70 (m,1H), 6.00-6.12 (m, 1H), 6.60-6.92 (m, 1H), 7.07 (br. s., 1H), 7.13 (br.s., 1H), 7.36 (br. s., 1H), 8.07 (br. s., 1H), 8.24 517.3 (br. s., 1H),8.53 (br. s., 1H), 9.89-10.08 (m, 1H) 72

CH2

1H NMR (400 MHz, DMSO- d6) Shift 1.58 (br. s., 1H), 1.95 (br. s., 1H),2.13-2.26 (m, 4H), 2.29 (br. s., 1H), 3.09 (s, 3H), 3.61-3.73 (m, 6H),3.79 (br. s., 1H), 3.96-4.28 (m, 2H), 4.53 (br. s., 1H), 4.70 (br. s.,1H), 5.50-5.70 (m, 1H), 6.00-6.12 (m, 1H), 6.61-6.89 (m, 1H), 7.05-7.16(m, 2H), 7.38 (br. s., 1H), 8.06 (br. s., 1H), 8.24 (s, 1H), 8.53 (br.s., 1H), 9.88-10.06 (m, 1H) 507.3 73

CH2

1H NMR (400 MHz, DMSO- d6) Shift 1.53-1.63 (m, 1H), 1.82-2.02 (m, 1H),2.22 (s, 6H), 3.52-3,60 (m, 2H), 3.66-4.27 (m, 7H), 4.34 (s, 3H),4.65-4.82 (m, 1H), 5.68 (br. s., 2H), 5.97-6.14 (m, 1H), 6.57-6.90 (m,1H), 6.98-7.17 (m, 2H), 7.30-7.44 (m, 1H), 8.10 (s, 1H), 8.24 (s, 1H),8.40-8.62 (m, 1H), 9.88-10.10 (m, 1H) 519.3 74

NH

1H NMR (400 MHz, DMSO- d6) Shift 1.58 (br. s., 1H), 1.95 (br. s., 1H),2.17 (br. s., 4H), 2.29 (br. s., 1H), 2.90 (d, J = 4.10 Hz, 8H), 3.07(br. s., 1H), 3.78 (br. s., 1H), 3.97-4.15 (m, 1H), 4.16-4.56 (m, 1H),4.75 (br. s., 1H), 5.50-5.73 (m, 1H), 6.01-6.12 (m, 1H), 6.84 (br. s.,1H), 7.21-7.40 (m, 3H), 8.05 (br. s., 1H), 8.21-8.30 (m, 3H), 8.57 (br.s., 1H), 9.81- 9.98 (m, 1H) 492.3 75

NH

1H NMR (400 MHz, DMSO) Shift 1.56 (br. s., 1H), 1.82 (br. s., 4H), 1.93(br. s., 1H), 2.17 (br. s., 4H), 2.29 (br. s., 1H), 3.78 (br. s., 1H),3.92- 4.14 (m, 1H), 4.49 (br. s., 1H), 4.72 (br. s., 1H), 5.51- 5.70 (m,1H), 6.00-5.12 (m, 1H), 6.50-6.88 (m, 1H), 7.25 (br. s., 1H), 7.34 (br.s., 1H), 7.40 (br. s., 1H), 8.00-8.12 (m, 2H), 8.21- 8.32 (m, 1H), 8.57(br. s., 1H), 9.82-9.98 (m, 1H) 518.3 76

CH2

1H-NMR (DMSO-D6) δ: 10.21 (s, 1H), 8.43 (br s, 1H), 8.31 (s, 1H), 8.21(br s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.24(dd, J = 8.3, 2.0 Hz, 1H), 6.88-6.72 (m, 1H), 6.19-6.07 (m, 1H),5.75-5.63 (m, 1H), 5.27-4.97 (m, 2H), 4.66- 4.30 (m, 2H), 3.79-3.25 (m,3H), 3.73 (s, 2H), 3.03 (s, 3H), 2.84 (s, 3H), 2.72- 2.54 (m, 1H). 529.4

TABLE 10 77

CH2

1H-NMR (DMSO-D6) δ: 10.33-10.21 (m, 1H), 8.62 (br s, 1H), 8.27 (s. 1H),8.16 (br s, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 6.0 Hz, 1H),6.88-6.70 (m, 1H), 6.20-6.06 (m, 1H), 5.75-5.62 (m, 1H), 5.36-5.17 (m,1H), 5.10-4.92 (m, 1H), 4.65-4.25 (m, 2H), 3.84-3.24 (m, 3H), 3.71 (s,2H), 3.04 (s, 3H), 2.85 (s, 3H), 2.77-2.58 (m, 1H), 2.13 (s, 3H), 2.10(s, 3H). 523.5 78

CH2

1H NMR (DMSO-D6) δ: 10.19-10.16 (m, 1H), 8.41 (s, 1H), 8.29 (s, 1H),8.20 (s, 1H), 7.85-7.78 (m, 1H), 7.45-7.42 (m, 1H), 7.27- 7.22 (m, 1H),6.73-6.58 (m, 1H), 623-6.11 (m, 1H), 5.75-5.64 (m, 1H), 5.47-5.36 (m,1H), 4.42-3.93 (m, 3H), 3.73 (s, 2H), 3.03(s, 3H), 2.84 (s, 3H),2.87-2.87 (m, 1H), 2.32- 2.24 (m, 1H), 1.30 (d, J = 6.1 Hz, 3H). 511.579

CH2

1H-NMR (DMSO-D6) δ: 1.30-1.36 (m, 3H), 2.10 (s, 3H), 2.13 (s, 3H),2.30-2.39 (m, 1H), 2.61-2.89 (m, 1H), 2.80 (s, 3H), 3.04 (s, 3H), 3.71(s, 2H), 3.34-4.41 (m, 3H), 5.30-5.46 (m, 1H), 5.63-5.74 (m, 1H),6.12-6.24 (m, 1H), 8.57-8.71 (m, 1H), 6.94-6.99 (m, 1H), 7.11-7.16 (m,1H), 8.11 (br s, 1H), 8.27 (s, 1H), 8.60 (br s, 1H), 10.25-10.29 (m,1H). 505.2

Test Example 1 Measurement of Inhibitory Effect on HER2-PhosphorylatingActivity (In Vitro)

For setting the conditions for the method for measuring the in vitroinhibitory activity of a compound against HER2-phosphorylating activity,ProfilerPro Peptide 22 from PerkinElmer Inc. was used as a substrate onthe basis of the report (PLoS One, 6 (7), e21487, 2011) on HER2 kinasereaction using, as a substrate, a peptide having the same sequence(5-FAM-EEPLYWSFPAKKK-CONH2) as that of ProfilerPro Peptide 22. Thepurified recombinant human HER2 protein used in the test was purchasedfrom Carna Biosciences, Inc.

For the inhibitory activity measurement of each compound, the synthesisexample compound was first serially diluted with dimethyl sulfoxide(DMSO). Next, the HER2 protein, the substrate peptide (finalconcentration: 0.5 μM), manganese chloride (final concentration: 10 mM),ATP (final concentration: 6 μM), and the solution of the synthesisexample compound in DMSO (final concentration of DMSO: 5%) were addedinto a buffer solution for kinase reaction (15 mM Tris (pH 7.5), 2 mMdithiothreitol, and 0.01% Tween 20), and the mixture was incubated at25° C. for 40 minutes for kinase reaction. The reaction was terminatedby adding EDTA (final concentration: 30 mM) thereto. Finally, theunphosphorylated substrate peptide (S) and the phosphorylated peptide(P) were separated and detected by microcapillary electrophoresis usingLabChip® EZ Reader II (PerkinElmer Inc.). The amount of phosphorylationreaction was determined from the respective peak heights of S and P. Thecompound concentration which can suppress the phosphorylation reactionby 50% was defined as an IC50 value (nM).

The results are shown in the table below.

TABLE 11 HER2 inhibitory activity IC50 Synthesis value Example No. (nM)1 10 2 8.3 3 7.1 4 12 5 11 6 4.0 7 7.1 8 9.2 9 10 10 13 11 7.5 12 7.5 1313 14 4.6 15 8.6 16 10 17 5.9 18 4.4 19 10 20 13 21 26 22 48 23 20 24 3825 21 26 7.0 27 3.9 28 46 29 6.5 30 8.7 31 13 32 9.3 33 6.0 34 11 35 1636 5.4 37 11 38 10 39 22 40 23 41 18 42 18 43 14 44 46 45 28 46 44 47 5248 8.5 49 20 50 2.9 51 17 52 13 53 14 54 31 55 49 56 22 57 31 58 11 5945 60 20 61 13 62 59 63 38 64 49 65 20 66 19 67 72 68 7.7 69 9.2 70 1071 11 72 11 73 74 74 17 75 16 76 5.0 77 13 78 14 79 17

The results of this test demonstrated that the pyrazolo[3,4-d]pyrimidinecompound of formula (I) has HER2 inhibitory activity in vitro.

Test Example 2 Measurement Test (In Vitro) of Cytostatic ActivityAgainst HER2-Expressing Cell Line (NCI-N87) and EGFR-Expressing CellLine (A431) and Comparison of Selectivity Thereof

HER2-overexpressing human stomach cancer cell line NCI-N87 cells weresuspended in RPMI1640 medium (manufactured by Life Technologies Corp.)containing 10% fetal bovine serum. Meanwhile, EGFR-overexpressing highlyactivated human epithelioid cancer cell line A431 cells were suspendedin DMEM, high glucose medium (manufactured by Life Technologies Corp.)containing 10% fetal bovine serum. Subsequently, each cell suspensionwas inoculated to each well of a 384-well flat-bottom microplate andcultured at 37° C. for 1 day in an incubator containing 5% carbondioxide. The synthesis example compound was dissolved in DMSO, and thetest compound was diluted into a concentration of 500 times the finalconcentration using DMSO. The solution of the test compound in DMSO wasdiluted with the medium used in the suspension of each cell line, andthis solution was added at a final concentration of DMSO of 0.2% to eachwell of the cell culture plate, followed by further culture at 37° C.for 3 days in an incubator containing 5% carbon dioxide. The number ofcells after the 3-day culture in the presence of the compound wascounted using CellTiter-Glo (manufactured by Promega Corp.) on the basisof the protocol recommended by Promega Corp. The rate of inhibition ofgrowth was calculated according to the expression given below todetermine the concentration at which the test compound inhibited thecell growth by 50% (IC50 (nM)).

The results are shown in the table below.

Rate of growth inhibition (%)=(C−T)/(C)×100

T: Luminescence intensity of a well supplemented with the test compoundC: Luminescence intensity of a well not supplemented with the testcompound

TABLE 12 Synthesis NCI-N87 cell growth A431 cell growth Exampleinhibitory activity inhibitory activity No. IC50 value (nM) IC50 value(nM) 1 19 2319 2 45 2666 3 27 3373 4 12 2607 5 6 1233 6 7 1289 7 12 28058 8 955 9 13 2446 10 6 1414 11 4 961 12 3 340 13 5 1211 14 4 486 15 202270 16 27 3456 17 5 910 18 12 1058 19 12 1261 26 5 984 27 5 1508 29 462223 30 23 2349 32 7 1921 33 3 263 34 32 2206 35 8 2607 38 8 1040 39 121799 48 4 314 50 4 277 63 11 1669 56 31 2613 57 44 3389 58 8 681 60 505256 61 10 1099 66 50 6040 71 26 2115 72 5 1172 74 18 8075 76 S 2074 7719 2455 78 41 5450 79 21 6942

The test results demonstrated that the pyrazolo[3,4-d]pyrimidinecompound of formula (I) showed excellent cytostatic activity against theHER2-overexpressing cell line NCI-N87 and that thepyrazolo[3,4-d]pyrimidine compound of formula (I) not only inhibits HER2kinase but has excellent cytostatic activity against the cells.

In addition, the results also demonstrated that thepyrazolo[3,4-d]pyrimidine compound of formula (I) also has excellentHER2 kinase inhibition selectivity over EGFR kinase.

Test Example 3 Measurement Test (In Vitro) of Cytostatic ActivityAgainst HER2-Expressing Cell Line (SK-BR-3)

HER2-overexpressing human breast cancer cell line SK-BR-3 cells weresuspended in McCoy's 5a medium (manufactured by Life Technologies Corp.)containing 10% fetal bovine serum. The cell suspension was inoculated toeach well of a 384-well flat-bottom microplate and cultured at 37° C.for 1 day in an incubator containing 5% carbon dioxide. The synthesisexample compound was dissolved in DMSO, and the test compound wasdiluted into 500 times as high concentration as the final concentrationusing DMSO. The solution of the test compound in DMSO was diluted withthe medium used in the suspension of each cell line, and this solutionwas added at a final concentration of DMSO of 0.2% to each well of thecell culture plate, followed by further culture at 37° C. for 3 days inan incubator containing 5% carbon dioxide. The number of cells after the3-day culture in the presence of the compound was counted usingCellTiter-Glo (manufactured by Promega Corp.) on the basis of theprotocol recommended by Promega Corp. The rate of inhibition of growthwas calculated according to the expression given above to determine theconcentration at which the test compound inhibited the cell growth by50% (IC50 (nM)).

TABLE 13 Synthesis SK-BR-3 cell growth inhibitory Example No. activityIC50 value (nM) 1 20 2 30 3 9 4 16 5 11 6 14 7 31 10 21 11 12 13 9 14 1015 30 16 40 17 13 19 24 26 8 27 9 29 44 30 39 32 14 33 9 36 12 38 12 3924 53 18 58 11 60 39 61 15 66 29 71 19 72 16 76 8 77 12 78 36 79 13

The results of this test demonstrated that the pyrazolo[3,4-d]pyrimidinecompound of formula (I) also has excellent cytostatic activity againstthe HER2-overexpressing human breast cancer cell line.

EXAMPLES

In Examples below, Compound 1 represents(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamidewhich is the compound of Synthesis Example 13.

Example 1 Combination of Compound 1 and Trastuzumab (In Vivo)

Human stomach cancer cells 4-1st were obtained as fragment tumor fromthe Central Institute for Experimental Animals. Tumor was excisedapproximately one month after subcutaneous transplantation and passagein a nude mouse (BALB/cAJcl-nu/nu, CLEA Japan, Inc.), and prepared intoa fragment of approximately 2 mm square. A transplantation needle waspacked with one fragment and subcutaneously inserted from around theright most distal rib of a nude mouse. After entrance to the right back,the inner cylinder was pushed to eject and transplant the tumor.Electronic calipers were used in tumor size measurement, and the majoraxis and the minor axis of the tumor of each mouse were measured bysandwiching the tumor between the measuring surfaces of the electroniccalipers (Days 0, 4, 8, 11 and 15). A tumor volume (TV) was calculatedfrom the major axis and the minor axis. A relative tumor volume (RTV)and a rate of change in relative tumor volume (T/C) were calculated fromthe calculated tumor volume. TV, RTV, and T/C were calculated accordingto the following expressions.

Tumor volume TV (mm³)=(Major axis, mm)×(Minor axis, mm)×(Minor axis,mm)/2

Relative tumor volume RTV=(TV on the day of measurement)/(TV on the dayof grouping)

Rate of change in relative tumor volume T/C (%)=(Average RTV of anadministration group)/(Average RTV of a control group)×100

An electronic balance for animals was used in body weight measurement. Arate of change in body weight on day n (BWCn) was calculated from thebody weight on day n (BWn) according to the following expression.

Rate of change in body weight BWCn (%)=(BWn−BW0)/BW0×100

Nude mice whose TV reached 100 to 300 mm³ were selected, and six animalswere assigned to each group by the equal number method (MiSTAT (ver.2.0)) such that average TV was equalized among groups. Trastuzumab(Herceptin, Roche) was injected intravenously to the tail vein once aday on Days 1 and 8. Compound 1 was orally administered once a day forconsecutive days from Days 1 to 14.

The presence or absence of an effect of combined use and toxicity weredetermined from tumor sizes and body weights on the day of grouping (Day0) and the day of evaluation (Day 15). During the administration period,the body weights were measured every day for the calculation of theamount of a dosing solution.

The results are shown in the table below and FIG. 1.

TABLE 14 RTV BWC (%) Dose Schedule Day 15 T/C Day 15 Group (mg/kg/day)(day) Route Mean + SD (%) Mean Control — 1-14 p.o. 13.38 ± 1.14 100.03.42 Trastuzumab 20 1, 8 i.v.  5.43 ± 0.46 ** 40.5 5.18 Compound 1 7.51-14 p.o.  6.55 ± 0.37 ** 49.0 5.06 Compound 1 15 1-14 p.o.  3.77 ± 0.32** 28.2 0.82 Trastuzumab/ 20/7.5 1, 8/1-14 i.v./p.o.  2.38 ± 0.30**^($$##) 17.8 2.10 Compound 1 Trastuzumab/ 20/15  1, 8/1-14 i.v./.p.o. 0.07 ± 0.01 **^($$##) 0.5 0.50 Compound 1 **: p < 0.01 withAspin-Welch's t-test as compared with the control group. ^($$): p < 0.01with Aspin-Welch's t-test as compared with the Trastuzumab group ^(##):p < 0.01 with Aspin-Welch's t-test as compared with the Compound 1 SD:standard deviation

As a result of analyzing RTV on Day 15 of each group by theAspin-Welch's t-test, RTV was found to be significantly lower in thesingle-drug group of the trastuzumab group or the Compound 1 group, andthe combined administration group of the trastuzumab/Compound 1 groupthan in the control group. Furthermore, RTV of the combinedadministration group of the trastuzumab/Compound 1 group was found to besignificantly lower than that of the single-drug group of thetrastuzumab group or the Compound 1 group. Moreover, the average rate ofchange in body weight of the combined administration group on the day ofevaluation involved no enhancement of toxicity as compared with thesingle-drug group of the trastuzumab group or the Compound 1 group.

Example 2 Combination of Compound 1 and Trastuzumab Emtansine (In Vivo)

The experiment was conducted in the same way as in Example 1 except thattrastuzumab emtansine (Kadcyla, Roche) was used instead of trastuzumaband administered from the tail vein once a day on Day 1.

The results are shown in the table below and FIG. 1.

TABLE 15 RTV BWC (%) Dose Schedule Day 15 T/C Day 15 Group (mg/kg/day)(day) Route Mean ± SD (%) Mean Control — 1-14 p.o. 11.48 ± 1.23 100.06.68 Trastuzumab emtansine 7.5 1 iv.  2.47 ± 0.75 ** 21.5 3.31 Compound1 7.5 1-14 p.o.  5.92 ± 0.38 ** 51.6 7.41 Compound 1 15 1-14 p.o.  3.58± 0.41 ** 31.2 3.63 Trastuzumab emtansine/ 7.5/7.5 1/1-14 iv./p.o,  0.18± 0.03 **^($$##) 1.6 2.89 Compound 1 Trastuzumab emtansine/ 7.5/15 1/1-14 iv./p.o.  0.05 ± 0.01 **^($$##) 0.4 3.99 Compound 1 **: p < 0.01with Aspin-Welch's t-test as compared with the control group. ^($$): p <0.01 with Aspin-Welch's t-test as compared with the Trastuzumabemtansine group ^(##): p < 0.01 with Aspin-Welch's t-test as comparedwith the Compound 1 SD: standard deviation

As a result of analyzing RTV on Day 15 of each group by theAspin-Welch's t-test, RTV was found to be significantly lower in thesingle-drug group of the trastuzumab emtansine group or the Compound 1group, and the combined administration group of the trastuzumabemtansine/Compound 1 group than in the control group. Furthermore, RTVof the combined administration group of the trastuzumabemtansine/Compound 1 group was found to be significantly lower than thatof the single-drug group of the trastuzumab emtansine group or theCompound 1 group. Moreover, the average rate of change in body weight ofthe combined administration group on the day of evaluation involved noenhancement of toxicity as compared with the single-drug group of thetrastuzumab emtansine group or the Compound 1 group.

Example 3 Combination of Compound 1 and Capecitabine (In Vivo)

A human stomach cancer cell line NCI-N87 was obtained from American TypeCulture Collection (ATCC). The cell line was cultured at 37° C. in a 5%CO₂ incubator using RPMI-1640 (containing 4.5 g/L glucose, 10 mM HEPES,and 1 mM sodium pyruvate) (Wako Pure Chemical Industries, Ltd.) mediumcontaining 10% fetal bovine serum (FBS).

The NCI-N87 cells were resuspended at a concentration of 8×10⁷ cells/mLin PBS.

The cell suspension was subcutaneously transplanted in an amount of8×10⁶ cells/0.1 mL to the right chest of a 6-week-old nude mouse(BALB/cAJcl-nu/nu, CLEA Japan, Inc.) using a 1 mL syringe for tuberculinand a 25 G injection needle.

Electronic calipers were used in tumor size measurement. The major axisand the minor axis of tumor were measured, and TV, RTV, and T/C werecalculated according to the expressions described above.

An electronic balance for animals was used in body weight measurement. Arate of change in body weight on day n (BWCn) was calculated from thebody weight on day n (BWn) according to the expression described above.

Nude mice whose TV reached 100 to 300 mm³ were selected, and fiveanimals were assigned to each group by the equal number method (MiSTAT(ver. 2.0)) such that average TV was equalized among groups.Capecitabine (Xeloda, Roche) and Compound 1 were orally administeredonce a day for consecutive days from Days 1 to 14.

The presence or absence of an effect of combined use and toxicity weredetermined from tumor sizes and body weights on the day of grouping (Day0) and the day of evaluation (Day 15). During the administration period,the body weights were measured every day for the calculation of theamount of a dosing solution.

The results are shown in the table below and FIGS. 3 and 4.

TABLE 16 RTV BWC (%) Dose Schedule Day 15 T/C Day 15 Group (mg/kg/day)(day) Route Mean ± SD (%) Mean Control — 1-14 p.o. 4.15 ± 0.49 100.08.94 Compound 1 15 1-14 p.o. 1.69 ± 0.39 *** 40.8 8.35 Compound 1 301-14 p.o. 0.67 ± 0.15 *** 16.3 6.43 Capecitabine 359 1-14 p.o. 1.26 ±0.21 *** 30.4 7.51 Capecitabine 809 1-14 p.o. 1.12 ± 0.18 *** 27.1 −5.30Compound 1/Capecitabine 15/359 1-14/1-14 p.o./p.o. 0.63 ± 0.08***^($$$###) 15.3 2.63 Compound 1/Capecitabine 30/359 1-14/1-14p.o./p.o. 0.35 ± 0.11 ***^($$$##) 8.4 1.52 Compound 1/Capecitabine15/809 1-14/1-14 p.o./p.o. 0.42 ± 0.10 ***^($$$###) 10.2 −0.20 Compound1/Capecitabine 30/809 1-14/1-14 p.o./p.o. 0.32 ± 0.06 ***^($$$###) 7.82.43 ***: p < 0.001 with Dunnett's test as compared with the controlgroup. ^($$$): p < 0.001 with Dunnett's test as compared with theCapecitabine group ^(##): p < 0.01 with Dunnett's test as compared withthe Compound 1 ^(###): p < 0.001 with Dunnett's test as compared withthe Compound 1 SD: standard deviation

As a result of analyzing RTV on Day 15 of each group by the Dunnett'stest, RTV was found to be significantly lower in the single-drug groupof the capecitabine group or the Compound 1 group, and the combinedadministration group of the Compound 1/capecitabine group than in thecontrol group. Furthermore, RTV of the combined administration group ofthe Compound 1/capecitabine group was found to be significantly lowerthan that of the single-drug group of the capecitabine group or theCompound 1 group. Moreover, the average rate of change in body weight ofthe combined administration group on the day of evaluation involved noenhancement of toxicity as compared with the single-drug group of thecapecitabine group or the Compound 1 group.

Example 4 Combination of Compound 1 and Other Antitumor Agents (InVitro)

The supplier of each reagent, the suppliers of tumor cell lines, themedia used, and the numbers of cells to be inoculated are shown in thetables below.

TABLE 17 Reagent Supplier Fetal bovine serum (FBS) MP Biomedicals, LLC,or Bovogen Biologicals Human epithelial growth factor R&D Systems, Inc.(hEGF) Leibovitz's L-15 medium Thermo Fisher Scientific, Inc. Dulbecco'smodified eagle's Wako Pure Chemical Industries, Ltd. medium (DMEM) ATCCformulated RPIVH-1640 Wako Pure Chemical Industries, Ltd. medium McCoy's5A medium Thermo Fisher Scientific, Inc. AZD8055 Cayman Chemical CompanyEverolirnus Cayman Chemical Company Dactohsib Cayman Chemical CompanyBuparlisib Cayman Chemical Company Taselisib Selleck ChemicalsPalbociclib Cayman Chemical Company 5-Fluorouracil Wako Pure ChemicalIndustries, Ltd. 5-Fluoro-2′-deoxyuridine Wako Pure Chemical Industries,Ltd. (FdUrd) Paclitaxel Wake Pure Chemical Industries, Ltd. CisplatinBristol-Myers Squibb Company Trifluridine (trifluorothyraidine) YukiGosei Kogyo Co., Ltd.

TABLE 18 The number Tumor of cells to be cell line Supplier of cellinoculated (origin) line Medium per well UACC-812 ATCC Leibovitz's L-15500 (human breast medium containing cancer) 20% FBS and 20 ng/mL hEGFMDA-MB-453 ATCC Leibovitz's L-15 500 (human breast medium containingcancer) 10% FBS KPL-4 Kawasaki Medical DMEM containing 250 (human breastSchool (Junichi 10% FBS cancer) Kurehayashi) NCI-N87 ATCC ATCCformulated 250 (human RPMI-1640 stomach medium containing cancer) 10%FBS SK-BR-3 Sumitomo Dainippon McCoy's 5A 250 (human breast Pharma Co.,Ltd. medium containing cancer) (formerly Dainippon 10% FBS Seiyaku K.K.)

The cells were inoculated at 20 μL/well to a 384-well culture plate(Corning Inc.). The plate inoculated with the cells were cultured in anincubator set to 37° C. and 5% CO₂. On the day following inoculation,each of mixed solutions of Compound 1 and each other antitumor agent atvarying combinations of concentrations was added to the cells.Specifically, 8 serial dilutions (including (0 nM)) of Compound 1 and 10serial dilutions (including (0 nM)) of the other antitumor agent wereprepared using Leibovitz's L-15 medium containing 10% FBS, or ATCCformulated RPMI-1640 medium containing 10% FBS. Drug solutions in whichequal amounts of the respective serial dilutions of the compounds weredispensed were mixed at a ratio of 1:1 to prepare a total of 80 mixeddrug solutions including every combination. The highest concentration(indicated by final concentration) and common ratio of each drug areshown in the table below. The highest concentration (indicated by finalconcentration) and common ratio of each drug are shown in the tablebelow. Each of these mixed drug solutions was added at 5 μL/well (N=4)to the plate, followed by further culture for 3 days under conditions of37° C. and 5% CO₂.

Three days later, CellTiter-Glo™ 2.0 Reagent (Promega Corporation) wasadded thereto at 25 μL/well, and chemiluminescence was measured using aplate reader (EnSpire® Multimode Plate Reader, PerkinElmer Japan Co.,Ltd.).

TABLE 19 Other antitumor agent Compound 1 Other Tumor Highest Highestantitumor cell concentration Common concentration Common agent line (nM)ratio (nM) ratio AZD8055 KPL-4 3000 3 3000 3 Everolimus MDA- 30000 103000 3 MB-453 Dactolisib UACC- 300 1.7 100 1.7 812 Buparlisib KPL-4 30003 3000 3 Taselisib KPL-4 3000 3 3000 3 Palbociclib KPL-4 15000 1.5 100003.4 5- NCI- 1000000 3 100 3 Fluorouracil N87 FdUrd NCI- 1000000 3 100 3N87 Paclitaxel KPL-4 50 3 50000 3 NCI- 50 3 100 3 N87 Gemcitabine NCI-1000 3 100 3 N87 Cisplatin SK- 167000 3 100 3 BR-3 NCI- 167000 3 100 3N87 Trifluridine NCI- 1000000 3 100 3 N87

The enhancement of an effect by combined use of the drugs was evaluatedaccording to a method known in the art (a method described in TrendsPharmacol. Sci., 4, 450-454, 1983 or Pharmacol Rev., 58 (3), 621-81,2006).

An average value from 4 wells of each combination was calculated fromthe obtained data, and a cell survival rate normalized against a controlsupplemented with a medium containing a vehicle was calculated. A Fa(fraction of affect) value was calculated by subtracting the cellsurvival rate from 1. Median effect analysis software CalcuSyn 2.0(CalcuSyn, Inc.) for calculation by the median effect method was used inthe calculation of CI.

As for the combinations of the concentrations of both the drugs forcalculating CI, a Fa value closer to 1 presumably means a concentrationrange in which one of the drugs had too strong an effect, and a Fa valuecloser to 0 presumably means a concentration range in which any of thedrugs had too weak an effect. Since these concentration ranges were notappropriate for the discussion of a synergistic effect, combinationsthat satisfied 0.2 S Fa 0.8 were extracted from the combinations of therespective concentrations of Compound 1 and the other antitumor agent onthe basis of the calculated Fa value.

An effect of combined use was determined as shown in the table below(Pharmacol Rev., 58 (3), p. 621-81, 2006).

TABLE 20 Range of CI (upper limit) Description 0.1 Very strongsynergistic effect 0.3 Strong synergistic effect 0.7 Synergistic effect0.85 Moderate synergistic effect 0.9 Slight synergistic effect 1.0Nearly additive 1.2 Slight antagonistic effect 1.45 Moderateantagonistic effect 3.3 Antagonistic effect 10 Strong antagonisticeffect >10 Very strong antagonistic effect

The results are shown in the tables below.

TABLE 21 Other antitumor agent: AZD8055, Cell line: KPL-4 Compound OtherCombined use ratio 1 antitumor (Compound 1:other (nM) agent (nM) Fa CIantitumor agent) 4.12 17.0 0.67 0.43 1:9.0 12.3 12.3 0.54 0.70 1:1.012.3 37.0 0.75 0.14 1:3.0 12.3 111 0.78 0.26 1:9.0 37.0 1.37 0.44 0.341:0.037 37.0 4.12 0.49 0.44 1:0.11 37.0 12.3 0.67 0.14 1:0.33 37.0 37.00.73 0.19 1:1.0 111 4.12 0.60 0.12 1:0.037 111 12.3 0.73 0.063 1:0.11111 37.0 0.78 0.087 1:0.33 333 12.3 0.72 0.074 1:0.037 113 37.0 0.790.073 1:0.11 1000 37.0 0.72 0.22 1:0.037

TABLE 22 Other antitumor agent: Everolimus, Cell line: MDA-MB-453Compound Other Combined use ratio 1 antitumor (Compound 1:other (nM)agent (nM) Fa CI antitumor agent) 37.0 3.00 0.71 0.071 1:0.081 37.0 30.00.71 0.21 1:0.81 111 30.0 0.76 0.15 1:0.27 111 300 0.79 0.50 1:2.7 3330.300 0.74 0.30 1:0.00090 333 3.00 0.76 0.24 1:0.0090 333 30.0 0.78 0.231:0.090 1000 3.00 0.76 0.70 1:0.0030 1000 30.0 0.79 0.51 1:0.030

TABLE 23 Other antitumor agent: Dactolisib, Cell line: UACC-812 CompoundOther Combined use ratio 1 antitumor (Compound 1:other (nM) agent (nM)Fa CI antitumor agent) 4.14 35.9 0.26 0.79 1:8.7 7.04 61.1 0.51 0.461:8.7 12.0 21.1 0.31 0.70 1:1.8 12.0 35.9 0.46 0.48 1:3.0 12.0 61.1 0.550.46 1:5.1 12.0 104 0.68 0.39 1:8.7 20.4 21.1 0.42 0.61 1:1.0 20.4 35.90.51 0.53 1:1.8 20.4 61.1 0.63 0.41 1:3.0 20.4 104 0.73 0.36 1:5.1 20.4176 0.79 0.38 1:8.7 34.6 21.1 0.51 0.66 1:0.61 34.6 35.9 0.54 0.65 1:1.034.6 61.1 0.65 0,51 1:1.8 34.6 104 0.75 0.41 1:3.0 58.8 61.1 0.71 0.551:1.0 58.8 104 0.77 0.49 1:1.8 100 61.1 0.78 0.59 1:0.61

TABLE 24 Other antitumor agent: Buparlisib, Cell line: KPL-4 CompoundOther Combined use ratio 1 antitumor (Compound 1:other (nM) agent (nM)Fa CI antitumor agent) 37.0 333 0.33 0.72 1:9.0 111 333 0.44 0.59 1:3.0333 333 0.50 0.58 1:1.0 1000 333 0.53 0.66 1:0.33 3000 333 0.58 0.761:0.11

TABLE 25 Other antitumor agent: Taselisib, Cell line: KPL-4 CompoundOther Combined use ratio 1 antitumor (Compound 1:other (nM) agent (nM)Fa CI antitumor agent) 4.12 37.0 0.76 0.0040 1:9.0 12.3 12.3 0.72 0.0131:1.0 12.3 37.0 0.79 0.0010 1:3.0 37.0 12.3 0.71 0.024 1:0.33 37.0 37.00.79 0.0010 1:1.0 37.0 111 0.78 0.0030 1:3.0 37.0 333 0.78 0.010 1:9.0111 12.3 0.65 0.51 1:0.11 111 37.0 0.78 0.0020 1:0.33 111 111 0.790.0020 1:1.0 333 333 0.79 0.0060 1:1.0 333 1000 0.77 0.058 1:3.0 3333000 0.77 0.17 1:9.0 1000 37.0 0.75 0.016 1:0.037 1000 111 0.77 0.0111:0.11 1000 333 0.75 0.072 1:0.33 1000 1000 0.75 0.20 1:1.0 3000 1110.79 0.011 1:0.037 3000 333 0.77 0.034 1:0.11 3000 1000 0.76 0.12 1:0.333000 3000 0.77 0.19 1:1.0

TABLE 26 Other antitumor agent: Palbociclib, Cell line: KPL-4 CompoundOther Combined use ratio 1 antitumor (Compound 1:other (nM) agent (nM)Fa CI antitumor agent) 22.0 878 0.27 0.15 1:40 22.0 1975 0.35 0.21 1:9074.8 878 0.50 0.062 1:12 74.8 1975 0.55 0.091 1:26 74.8 2963 0.51 0.161:40 254 2963 0.62 0.12 1:12 254 4444 0.62 0.16 1:17 254 6667 0.68 0.171:26 865 1975 0.58 0.19 1:2.3 865 2963 0.61 0.19 1:3.4 865 4444 0.580.27 1:5.1 2941 4444 0.64 0.39 1:1.5

TABLE 27 Other antitumor agent: 5-Fluorouracil, Cell line: NCI-N87Compound Other Combined use ratio 1 antitumor (Compound 1:other (nM)agent (nM) Fa CI antitumor agent) 0.137 12346 0.41 0.56 1:90000 0.4124115 0.30 0.55 1:10000 0.412 12346 0.39 0.60 1:30000 1.24 1372 0.22 0.491:1111 1.24 4115 0.34 0.34 1:3333 3.70 1372 0.31 0.74 1:370 3.70 41150.46 0.46 1:1111 3.70 12346 0.47 0.54 1:3333 3.70 37037 0.54 0.611:10000 3.70 111111 0.63 0.63 1:30000 3.70 333333 0.74 0.38 1:90000 11.14115 0.63 0.63 1:370 11.1 12346 0.66 0.59 1:1111 11.1 37037 0.69 0.581:3333 11.1 111111 0.77 0.39 1:10000

TABLE 28 Other antitumor agent: FdUrd, Cell line: NCI-N87 Compound OtherCombined use ratio 1 antitumor (Compound 1:other (nM) agent (nM) Fa CIantitumor agent) 0.412 4115 0.25 0.39 1:10000 0.412 12346 0.30 0.461:30000 0.412 37037 0.41 0.63 1:90000 1.24 12346 0.33 0.58 1:10000 1.2437037 0.39 0.70 1:30000 1.24 111111 0.54 0.39 1:90000 3.70 4115 0.390.82 1:1111 3.70 12346 0.47 0.58 1:3333 3.70 37037 0.56 0.41 1:100003.70 111111 0.57 0.60 1:30000 3.70 333333 0.62 0.47 1:90000 11.1 41150.61 0.79 1:370 11.1 12346 0.68 0.55 1:1111 11.1 37037 0.67 0.58 1:333311.1 111111 0.73 0.46 1:10000 11.1 333333 0.75 0.52 1:30000 11.1 10000000.79 0.31 1:90000 33.3 12346 0.80 0.76 1:370

TABLE 29 Other antitumor agent: Paclitaxel, Cell line: KPL-4 CompoundOther Combined use ratio 1 antitumor (Compound 1:other (nM) agent (nM)Fa CI antitumor agent) 68.6 0.00762 0.32 0.15 1:0.00011 68.6 0.0229 0.440.03 1:0.00033 206 0.617 0.33 0.50 1:0.0030 206 1.85 0.52 0.30 1:0.0090617 1.85 0.54 0.31 1:0.0030 1852 1.85 0.55 0.36 1:0.0010 5556 1.85 0.570.48 1:0.00033 5556 5.56 0.78 0.44 1:0.0010 16667 1.85 0.66 0.381:0.00011 50000 1.85 0.86 0.13 1:0.000037

TABLE 30 Other antitumor agent: Paclitaxel,Cell line: NCI-N87 CompoundOther Combined use ratio 1 antitumor (Compound 1:other (nM) agent (nM)Fa Cl antitumor agent) 1.24 5.56 0.50 0.45 1:4.5 3.70 1.85 0.41 0.841:0.50 3.70 5.56 0.64 0.42 1:1.5 3.70 16.7 0.71 0.55 1:4.5 11.1 0.2060.69 0.58 1:0.019 11.1 0.617 0.71 0.52 1:0.056 11.1 1.85 0.69 0.611:0.17 11.1 5.56 0.78 0.44 1:0.50

TABLE 31 Other antitumor agent: Gemcitabine, Cell line: NCI-N87 CompoundOther Combined use ratio 1 antitumor (Compound 1: other (nM) agent (nM)Fa CI antitumor agent) 0.137 1.37 0.69 0.03 1:10 0.137 4.12 0.68 0.371:30 0.137 12.3 0.73 0.01 1:90 0.41 4.12 0.68 0.21 1:10 0.41 12.3 0.690.23 1:30 0.41 37.0 0.71 0.02 1:90 1.24 1.37 0.70 0.07 1:1.1 1.24 4.120.71 0.06 1:3.3 1.24 12.3 0.77 0.04 1:10 1.24 37.0 0.73 0.05 1:30 1.24111 0.72 0.05 1:90 3.70 1.37 0.75 0.15 1:0.37 3.70 4.12 0.77 0.12 1:1.13.70 12.3 0.80 0.10 1:3.3 3.70 37.0 0.78 0.11 1:10 3.70 111 0.78 0.111:30 3.70 333 0.77 0.12 1:90

TABLE 32 Other antitumor agent: Cisplatin, Cell line: SK-BR-3 CompoundOther Combined use ratio 1 antitumor (Compound 1:other (nM) agent (nM)Fa CI antitumor agent) 0.412 6185 0.50 0.86 1:15030 1.24 6185 0.55 0.841:5010 3.70 687 0.22 0.78 1:186 3.70 2062 0.37 0.79 1:557 3.70 6185 0.650.63 1:1670

TABLE 33 Other antitumor agent: Cisplatin, Cell line: NCI-N87 CompoundOther Combined use ratio 1 antitumor (Compound 1:other (nM) agent (nM)Fa CI antitumor agent) 0.137 2062 0.39 0.62 1:15030 0.412 2062 0.36 0.641:5010 0.412 6185 0.55 0.57 1:15030 1.24 6185 0.60 0.40 1:5010 1.2418556 0.72 0.42 1:15030 3.70 2062 0.54 0.58 1:557 3.70 6185 0.67 0.381:1670 3.70 18556 0.75 0.42 1:5010 11.1 687 0.59 0.87 1:62 11.1 20620.64 0.71 1:186 11.1 6185 0.74 0.48 1:557 11.1 18556 0.77 0.50 1:167011.1 55667 0.79 0.90 1:5010 33.3 2062 0.80 0.73 I:62

TABLE 34 Other antitumor agent: Trifluridine, Cell line: NCI-N87Compound Other Combined use ratio 1 antitumor (Compound 1:other (nM)agent (nM) Fa CI antitumor agent) 0.137 12346 0.43 0.26 1:90000 0.4121372 0.26 0.35 1:3333 0.412 4115 0.26 0.65 1:10000 0.412 12346 0.41 0.381:30000 0.412 37037 0.54 0.34 1:90000 1.24 1372 0.24 0.78 1:1111 1.244115 0.31 0.71 1:3333 1.24 12346 0.48 0.37 1:10000 1.24 37037 0.55 0.411:30000 1.24 111111 0.59 0.68 1:90000 3.70 12346 0.57 0.51 1:3333 3.7037037 0.61 0.51 1:10000 3.70 111111 0.67 0.51 1:30000 11.1 4115 0.690.79 1:370 11.1 12346 0.77 0.51 1:1111 11.1 37037 0.74 0.63 1:3333 11.1111111 0.80 0.49 1:10000

These results demonstrated that combined use of Compound 1 with theantimetabolite 5-fluorouracil, 5-fluoro-2′-deoxyuridine (FdUrd),gemcitabine, or trifluridine (trifluorothymidine), the platinum-baseddrug cisplatin, the alkaloid-based drug paclitaxel, the PI3K/AKT/mTORsignaling pathway inhibitor AZD8055, everolimus, dactolisib, buparlisib,or taselisib, or the CDK4/6 inhibitor palbociclib synergisticallyenhances the antitumor effect.

1: A composition, comprising: a pyrazolo[3,4-d]pyrimidine compound offormula (I) or a salt thereof, and one or more other antitumor agent(s),

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group; Y represents—C(R₄)═C(R₅)(R₆); Z₁, Z₂, Z₃, and Z₄ are the same as or different fromeach other and each represent a hydrogen atom, a halogen atom, a cyanogroup, a C2-C6 alkenyl group, an optionally substituted C1-C6 alkoxygroup, an optionally substituted C1-C6 alkyl group, an optionallysubstituted amino group, an optionally substituted C3-C7 cycloalkylgroup, a C6-C14 aromatic hydrocarbon group, or a 4- to 14-memberedunsaturated heterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionallyform, together with the respective carbon atoms bonded thereto, abenzene ring or a 5- to 7-membered saturated or unsaturated heterocyclicring; W represents —CH₂—, an oxygen atom, or —NH—; n represents aninteger of from 0 to 2; R₁ represents an optionally substituted aminogroup; R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; and R₄, R₅, and R₆ are the same as or different from each otherand each represent a hydrogen atom or an optionally substituted C1-C6alkyl group. 2: The composition according to claim 1, wherein in thepyrazolo[3,4-d]pyrimidine compound of formula (I), X is a 4- to8-membered nitrogen-containing saturated heterocyclic group optionallyhaving a halogen atom or a C1-C6 alkyl group as a substituent; Y is—C(R₄)═C(R₅)(R₆); Z₁, Z₂, Z₃, and Z₄ are the same as or different fromeach other and each are a hydrogen atom, a halogen atom, a cyano group,a C2-C6 alkenyl group, an optionally halogen atom-substituted C1-C6alkoxy group, a C1-C6 alkyl group, an optionally C1-C6 alkylgroup-substituted amino group, a C3-C7 cycloalkyl group, or a monocyclic4- to 6-membered unsaturated heterocyclic group having one oxygen atom,or Z₁ and Z₂, or Z₃ and Z₄ optionally form, together with the respectivecarbon atoms bonded thereto, a benzene ring or a 5- to 7-memberedsaturated or unsaturated heterocyclic ring; W is —CH₂—, an oxygen atom,or —NH—; n is 0; R₁ is an amino group; R₂ and R₃ are the same as ordifferent from each other and each are a hydrogen atom, a C1-C6 alkoxygroup, a C1-C6 alkyl group, or a C6-C14 aromatic hydrocarbon group, orR₂ and R₃ optionally form, together with the nitrogen atom bondedthereto, an optionally hydroxyl group-substituted 4- to 8-memberednitrogen-containing saturated heterocyclic group; and R₄, R₅, and R₆ arethe same as or different from each other and each are a hydrogen atom oran optionally di(C1-C6 alkyl)amino group-substituted C1-C6 alkyl group.3: The composition according to claim 1, wherein in thepyrazolo[3,4-d]pyrimidine compound of formula (I), X is a pyrrolidinylgroup, a methylpyrrolidinyl group, a piperidinyl group, or afluoropiperidinyl group; Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group, a fluoromethoxy group, adifluoromethoxy group, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring; W is—CH₂—, an oxygen atom, or —NH—; n is 0; R₁ is an amino group; and R₂ andR₃ are the same as or different from each other and each are a methoxygroup, a methyl group, or a phenyl group, or R₂ and R₃ optionally form,together with the nitrogen atom bonded thereto, a hydroxyazetidinylgroup, a pyrrolidinyl group, or a piperidinyl group. 4: The compositionaccording to claim 1, wherein the pyrazolo[3,4-d]pyrimidine compound isa compound selected from the group consisting of (1) to (20): (1)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-bromo-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(2)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(3)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(4)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(5) (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(6)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(7)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(8)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide, (9)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(10) (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-5-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(11)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(12) (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide, (13)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide, (14)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(15)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-(difluoromethoxy)-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(16) (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-(fluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(17)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-bromo-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(18)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(19)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,and (20)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2,5-dichloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.5: The composition according to claim 1, wherein the other antitumoragent(s) is at least one selected from the group consisting of anantimetabolite, a molecular targeting drug, a platinum-based drug, andan alkaloid-based drug. 6: The composition according to claim 1, whereinthe other antitumor agent(s) is at least one selected from the groupconsisting of 5-fluorouracil (5-FU), 5-fluoro-2′-deoxyuridine (FdUrd),trifluridine, gemcitabine, capecitabine, trastuzumab, trastuzumabemtansine, AZD8055, everolimus, dactolisib, buparlisib, taselisib,palbociclib, cisplatin, and paclitaxel. 7: An agent that enhances anantitumor effect of other antitumor agent(s), the agent comprising apyrazolo[3,4-d]pyrimidine compound of formula (I) or a salt thereof asan active ingredient:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group; Y represents—C(R₄)═C(R₅)(R₆); Z₁, Z₂, Z₃, and Z₄ are the same as or different fromeach other and each represent a hydrogen atom, a halogen atom, a cyanogroup, a C2-C6 alkenyl group, an optionally substituted C1-C6 alkoxygroup, an optionally substituted C1-C6 alkyl group, an optionallysubstituted amino group, an optionally substituted C3-C7 cycloalkylgroup, a C6-C14 aromatic hydrocarbon group, or a 4- to 14-memberedunsaturated heterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionallyform, together with the respective carbon atoms bonded thereto, abenzene ring or a 5- to 7-membered saturated or unsaturated heterocyclicring; W represents —CH₂—, an oxygen atom, or —NH—; n represents aninteger of from 0 to 2; R₁ represents an optionally substituted aminogroup; R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; and R₄, R₅, and R₆ are the same as or different from each otherand each represent a hydrogen atom or an optionally substituted C1-C6alkyl group. 8: An antitumor agent for treating a cancer patient dosedwith other antitumor agent(s), the antitumor agent comprising apyrazolo[3,4-d]pyrimidine compound or a salt thereof, wherein thepyrazolo[3,4-d]pyrimidine compound is a compound of formula (I):

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group; Y represents—C(R₄)═C(R₅)(R₆); Z₁, Z₂, Z₃, and Z₄ are the same as or different fromeach other and each represent a hydrogen atom, a halogen atom, a cyanogroup, a C2-C6 alkenyl group, an optionally substituted C1-C6 alkoxygroup, an optionally substituted C1-C6 alkyl group, an optionallysubstituted amino group, an optionally substituted C3-C7 cycloalkylgroup, a C6-C14 aromatic hydrocarbon group, or a 4- to 14-memberedunsaturated heterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionallyform, together with the respective carbon atoms bonded thereto, abenzene ring or a 5- to 7-membered saturated or unsaturated heterocyclicring; W represents —CH₂—, an oxygen atom, or —NH—; n represents aninteger of from 0 to 2; R₁ represents an optionally substituted aminogroup; R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; and R₄, R₅, and R₆ are the same as or different from each otherand each represent a hydrogen atom or an optionally substituted C1-C6alkyl group. 9-27. (canceled) 28: A method for treating a tumor,comprising performing combined administration of apyrazolo[3,4-d]pyrimidine compound of formula (I) or a salt thereof andother antitumor agent(s) to a subject in need thereof:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group; Y represents—C(R₄)═C(R₅)(R₆); Z₁, Z₂, Z₃, and Z₄ are the same as or different fromeach other and each represent a hydrogen atom, a halogen atom, a cyanogroup, a C2-C6 alkenyl group, an optionally substituted C1-C6 alkoxygroup, an optionally substituted C1-C6 alkyl group, an optionallysubstituted amino group, an optionally substituted C3-C7 cycloalkylgroup, a C6-C14 aromatic hydrocarbon group, or a 4- to 14-memberedunsaturated heterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionallyform, together with the respective carbon atoms bonded thereto, abenzene ring or a 5- to 7-membered saturated or unsaturated heterocyclicring; W represents —CH₂—, an oxygen atom, or —NH—; n represents aninteger of from 0 to 2; R₁ represents an optionally substituted aminogroup; R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; and R₄, R₅, and R₆ are the same as or different from each otherand each represent a hydrogen atom or an optionally substituted C1-C6alkyl group. 29: The treatment method according to claim 28, wherein inthe pyrazolo[3,4-d]pyrimidine compound of formula (I), X is a 4- to8-membered nitrogen-containing saturated heterocyclic group optionallyhaving a halogen atom or a C1-C6 alkyl group as a substituent; Y is—C(R₄)═C(R₅)(R₆); Z₁, Z₂, Z₃, and Z₄ are the same as or different fromeach other and each are a hydrogen atom, a halogen atom, a cyano group,a C2-C6 alkenyl group, an optionally halogen atom-substituted C1-C6alkoxy group, a C1-C6 alkyl group, an optionally C1-C6 alkylgroup-substituted amino group, a C3-C7 cycloalkyl group, or a monocyclic4- to 6-membered unsaturated heterocyclic group having one oxygen atom,or Z₁ and Z₂, or Z₃ and Z₄ optionally form, together with the respectivecarbon atoms bonded thereto, a benzene ring or a 5- to 7-memberedsaturated or unsaturated heterocyclic ring; W is —CH₂—, an oxygen atom,or —NH—; n is 0; R₁ is an amino group; R₂ and R₃ are the same as ordifferent from each other and each are a hydrogen atom, a C1-C6 alkoxygroup, a C1-C6 alkyl group, or a C6-C14 aromatic hydrocarbon group, orR₂ and R₃ optionally form, together with the nitrogen atom bondedthereto, an optionally hydroxyl group-substituted 4- to 8-memberednitrogen-containing saturated heterocyclic group; and R₄, R₅, and R₆ arethe same as or different from each other and each are a hydrogen atom oran optionally di(C1-C6 alkyl)amino group-substituted C1-C6 alkyl group.30: The treatment method according to claim 28, wherein in thepyrazolo[3,4-d]pyrimidine compound of formula (I), X is a pyrrolidinylgroup, a methylpyrrolidinyl group, a piperidinyl group, or afluoropiperidinyl group; Y is

Z₁, Z₂, Z₃, and Z₄ are the same as or different from each other and eachare a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, acyano group, a vinyl group, a methoxy group, a fluoromethoxy group, adifluoromethoxy group, a methyl group, an ethyl group, a dimethylaminogroup, a cyclopropyl group, or a furyl group, or Z₁ and Z₂, or Z₃ and Z₄optionally form, together with the respective carbon atoms bondedthereto, a benzene ring, a pyridine ring, or a dioxolane ring; W is—CH₂—, an oxygen atom, or —NH—; n is 0; R₁ is an amino group; and R₂ andR₃ are the same as or different from each other and each are a methoxygroup, a methyl group, or a phenyl group, or R₂ and R₃ optionally form,together with the nitrogen atom bonded thereto, a hydroxyazetidinylgroup, a pyrrolidinyl group, or a piperidinyl group. 31: The treatmentmethod according to claim 28, wherein the pyrazolo[3,4-d]pyrimidinecompound is a compound selected from the group consisting of (1) to(20): (1)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-bromo-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide, (2)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(3) (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide, (4)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(5)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(6)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(7)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(8)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(9)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(10)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-5-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(11)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(3-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(12) (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(13)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(14) (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-3-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(15)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-(difluoromethoxy)-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(16)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(4-(2-(dimethylamino)-2-oxoethyl)-2-(fluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(17) (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-bromo-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(18)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2-chloro-4-(2-(dimethylamino)-2-oxoethyl)-5-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,(19)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(5-chloro-4-(2-(dimethylamino)-2-oxoethyl)-2-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide,and (20)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(2,5-dichloro-4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.32: The treatment method according to claim 28, wherein the otherantitumor agent(s) is at least one selected from the group consisting ofan antimetabolite, a molecular targeting drug, a platinum-based drug,and an alkaloid-based drug. 33: The treatment method according to claim28, wherein the other antitumor agent(s) is at least one selected fromthe group consisting of 5-fluorouracil (5-FU), 5-fluoro-2′-deoxyuridine(FdUrd), trifluridine, gemcitabine, capecitabine, trastuzumab,trastuzumab emtansine, AZD8055, everolimus, dactolisib, buparlisib,taselisib, palbociclib, cisplatin, and paclitaxel. 34: A method forenhancing an antitumor effect of other antitumor agent(s), comprisingadministering a pyrazolo[3,4-d]pyrimidine compound of formula (I) or asalt thereof to a subject in need thereof:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group; Y represents—C(R₄)═C(R₅)(R₆); Z₁, Z₂, Z₃, and Z₄ are the same as or different fromeach other and each represent a hydrogen atom, a halogen atom, a cyanogroup, a C2-C6 alkenyl group, an optionally substituted C1-C6 alkoxygroup, an optionally substituted C1-C6 alkyl group, an optionallysubstituted amino group, an optionally substituted C3-C7 cycloalkylgroup, a C6-C14 aromatic hydrocarbon group, or a 4- to 14-memberedunsaturated heterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionallyform, together with the respective carbon atoms bonded thereto, abenzene ring or a 5- to 7-membered saturated or unsaturated heterocyclicring; W represents —CH₂—, an oxygen atom, or —NH—; n represents aninteger of from 0 to 2; R₁ represents an optionally substituted aminogroup; R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; and R₄, R₅, and R₆ are the same as or different from each otherand each represent a hydrogen atom or an optionally substituted C1-C6alkyl group. 35: A method for treating a tumor in a cancer patient dosedwith other antitumor agent(s), comprising administering apyrazolo[3,4-d]pyrimidine compound of formula (I) or a salt thereof to asubject in need thereof:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group; Y represents—C(R₄)═C(R₅)(R₆); Z₁, Z₂, Z₃, and Z₄ are the same as or different fromeach other and each represent a hydrogen atom, a halogen atom, a cyanogroup, a C2-C6 alkenyl group, an optionally substituted C1-C6 alkoxygroup, an optionally substituted C1-C6 alkyl group, an optionallysubstituted amino group, an optionally substituted C3-C7 cycloalkylgroup, a C6-C14 aromatic hydrocarbon group, or a 4- to 14-memberedunsaturated heterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionallyform, together with the respective carbon atoms bonded thereto, abenzene ring or a 5- to 7-membered saturated or unsaturated heterocyclicring; W represents —CH₂—, an oxygen atom, or —NH—; n represents aninteger of from 0 to 2; R₁ represents an optionally substituted aminogroup; R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; and R₄, R₅, and R₆ are the same as or different from each otherand each represent a hydrogen atom or an optionally substituted C1-C6alkyl group. 36: A method for treating a tumor, comprising administeringa combination of pyrazolo[3,4-d]pyrimidine compound of formula (I) or asalt thereof and other antitumor agent(s) to a subject in need thereof:

wherein X represents an optionally substituted 4- to 10-memberednitrogen-containing saturated heterocyclic group; Y represents—C(R₄)═C(R₅)(R₆); Z₁, Z₂, Z₃, and Z₄ are the same as or different fromeach other and each represent a hydrogen atom, a halogen atom, a cyanogroup, a C2-C6 alkenyl group, an optionally substituted C1-C6 alkoxygroup, an optionally substituted C1-C6 alkyl group, an optionallysubstituted amino group, an optionally substituted C3-C7 cycloalkylgroup, a C6-C14 aromatic hydrocarbon group, or a 4- to 14-memberedunsaturated heterocyclic group, or Z₁ and Z₂, or Z₃ and Z₄ optionallyform, together with the respective carbon atoms bonded thereto, abenzene ring or a 5- to 7-membered saturated or unsaturated heterocyclicring; W represents —CH₂—, an oxygen atom, or —NH—; n represents aninteger of from 0 to 2; R₁ represents an optionally substituted aminogroup; R₂ and R₃ are the same as or different from each other and eachrepresent a hydrogen atom, an optionally substituted C1-C6 alkoxy group,an optionally substituted C1-C6 alkyl group, or an optionallysubstituted C6-C14 aromatic hydrocarbon group, or R₂ and R₃ optionallyform, together with the nitrogen atom bonded thereto, an optionallysubstituted 4- to 8-membered nitrogen-containing saturated heterocyclicgroup; and R₄, R₅, and R₆ are the same as or different from each otherand each represent a hydrogen atom or an optionally substituted C1-C6alkyl group.